Automated cell processing methods, modules, instruments, and systems

ABSTRACT

In an illustrative embodiment, automated multi-module cell editing instruments are provided to automate multiple edits into nucleic acid sequences inside one or more cells.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 62/527,339, entitled “Automated Editing of Nucleic Acids Withina Cell,” filed Jun. 30, 2017; U.S. Patent Application Ser. No.62/551,069, entitled “Electroporation Cuvettes for Automation,” filedAug. 28, 2017; U.S. Patent Application Ser. No. 62/566,374, entitled“Electroporation Device,” filed Sep. 30, 2017; U.S. Patent ApplicationSer. No. 62/566,375, entitled “Electroporation Device,” filed Sep. 30,2017; U.S. Patent Application Ser. No. 62/566,688, entitled“Introduction of Exogenous Materials into Cells,” filed Oct. 2, 2017;U.S. Patent Application Ser. No. 62/567,697, entitled “Automated NucleicAcid Assembly and Introduction of Nucleic Acids into Cells,” filed Oct.3, 2017; U.S. Patent Application Ser. No. 62/620,370, entitled“Automated Filtration and Manipulation of Viable Cells,” filed Jan. 22,2018; U.S. Patent Application Ser. No. 62/649,731, entitled “AutomatedControl of Cell Growth Rates for Induction and Transformation,” filedMar. 29, 2018; U.S. Patent Application Ser. No. 62/671,385, entitled“Automated Control of Cell Growth Rates for Induction andTransformation,” filed May 14, 2018; U.S. Patent Application Ser. No.62/648,130, entitled “Genomic Editing in Automated Systems,” filed Mar.26, 2018; U.S. Patent Application Ser. No. 62/657,651, entitled“Combination Reagent Cartridge and Electroporation Device,” filed Apr.13, 2018; U.S. Patent Application Ser. No. 62/657,654, entitled“Automated Cell Processing Systems Comprising Cartridges,” filed Apr.13, 2018; and U.S. Patent Application Ser. No. 62/689,068, entitled“Nucleic Acid Purification Protocol for Use in Automated Cell ProcessingSystems,” filed Jun. 20, 2018. All above identified applications arehereby incorporated by reference in their entireties for all purposes.

BACKGROUND

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

Genome editing with engineered nucleases is a method in which changes tonucleic acids are made in the genome of a living organism. Certainnucleases create site-specific double-strand breaks at target regions inthe genome, which can be repaired by nonhomologous end-joining orhomologous recombination, resulting in targeted edits. These methods,however, have not been compatible with automation due to lowefficiencies and challenges with cell transformation, growthmeasurement, and cell selection. Moreover, traditional benchtop devicesdo not necessarily scale and integrate well into an automated, modularsystem. Methods and systems to create edited cell populations thusremain cumbersome, and the challenges of introducing multiple rounds ofedits using recursive techniques has limited the nature and complexityof cell populations that can be created.

There is thus a need for automated instruments, systems and methods forintroducing assembled nucleic acids and other biological molecules intoliving cells in an automated fashion where the edited cells may be usedfor further experimentation outside of the automated instrument.

SUMMARY OF ILLUSTRATIVE EMBODIMENTS

In certain embodiments, automated methods are used for nuclease-directedgenome editing of one or more target genomic regions in multiple cells,the methods being performed in automated multi-module cell editinginstruments. These methods can be used to generate libraries of livingcells of interest with desired genomic changes. The automated methodscarried out using the automated multi-module cell editing instrumentsdescribed herein can be used with a variety of nuclease-directed genomeediting techniques, and can be used with or without use of one or moreselectable markers.

The present disclosure thus provides, in selected embodiments, modules,instruments, and systems for automated multi-module cell editing,including nuclease-directed genome editing. Other specific embodimentsof the automated multi-module cell editing instruments of the disclosureare designed for recursive genome editing, e.g., sequentiallyintroducing multiple edits into genomes inside one or more cells of acell population through two or more editing operations within theinstruments.

Thus, provided herein are embodiments of an automated multi-module cellediting instrument comprising: a housing configured to contain all orsome of the modules; a receptacle configured to receive cells; one ormore receptacles configured to receive nucleic acids; a transformationmodule configured to introduce the nucleic acids into the cells; arecovery module configured to allow the cells to recover after celltransformation in the transformation module; an editing moduleconfigured to allow the nucleic acids transformed into the cells to editnucleic acids in the cells; and a processor configured to operate theautomated multi-module cell editing instrument based on user inputand/or selection of an appropriate controller script.

In some aspects, the nucleic acids in the one or more receptaclescomprise a backbone and an editing cassette, and the automatedmulti-module cell editing instrument further comprises a nucleic acidassembly module. In some aspects, the nucleic acid assembly modulecomprises a magnet, and in some aspects, the nucleic acid assemblymodule is configured to perform assembly using a single isothermalreaction. In other aspects, the nucleic acid assembly module isconfigured to perform an amplification and/or ligation method.

In some aspects of the automated multi-module cell editing instrument,the editing module and the recovery module are combined.

In some aspects, the automated multi-module cell editing instrument mayfurther comprise a growth module configured to grow the cells, and insome implementations, the growth module measures optical density of thegrowing cells, either continuously or at intervals. In someimplementations, the processor is configured to adjust growth conditionsin the growth module such that the cells reach a target optical densityat a time requested by a user. Further, in some embodiments, the usermay be updated regarding growth process.

In some aspects, the automated multi-module cell editing instrumentcomprises a reagent cartridge where the receptacle configured to receivecells and the one or more receptacles configured to receive nucleicacids are contained within a reagent cartridge. Further, the reagentcartridge may also contain some or all reagents required for cellediting. In some implementations, the reagents contained within thereagent cartridge are locatable by a script read by the processor, andin some implementations, the reagent cartridge includes reagents and isprovided in a kit.

In some aspects, the transformation module of the automated multi-modulecell editing instrument comprises an electroporation device; and in someimplementations, the electroporation device is a flow-throughelectroporation device.

Some aspects of the automated multi-module cell editing instrumentfurther comprise a filtration module configured to exchange liquidsand/or concentrate the cells. In specific aspects, the filtration systemcan also be used to render the cells electrocompetent.

In other embodiments, an automated multi-module cell editing instrumentis provided, where the automated multi-module cell editing instrumentcomprises a housing configured to house some or all of the modules; areceptacle configured to receive cells; at least one receptacleconfigured to receive a nucleic acid backbone and an editing cassette; anucleic acid assembly module configured to a) assemble the backbone andediting cassette, and b) de-salt assembled nucleic acids after assembly;a growth module configured to grow the cells and measure optical density(OD) of the cells; a filtration module configured to concentrate thecells and render the cells electrocompetent; a transformation modulecomprising a flow-through electroporator to introduce the assemblednucleic acids into the cells; a combination recovery and editing moduleconfigured to allow the cells to recover after electroporation in thetransformation module and to allow the assembled nucleic acids to editnucleic acids in the cells; and a processor configured to operate theautomated multi-module cell editing instrument based on user inputand/or selection of an appropriate controller script.

In some implementations, the automated multi-module cell editinginstrument provides a reagent cartridge comprising a plurality ofreagent reservoirs, a flow-through electroporation device, and a scriptreadable by a processor for dispensing reagents located in the pluralityof reagent reservoirs and controlling the flow-through electroporationdevice.

In some aspects, the growth module includes a temperature-controlledrotating growth vial, a motor assembly to spin the vial, aspectrophotometer for measuring, e.g., OD in the vial, and a processorto accept input from a user and control the growth rate of the cells.The growth module may automatically measure the OD of the growing cellsin the rotating growth vial continuously or at set intervals, andcontrol the growth of the cells to a target OD and a target time asspecified by the user. That is, the methods and devices described hereinprovide a feedback loop that monitors cell growth in real time, andadjusts the temperature of the rotating growth vial in real time toreach the target OD at a target time specified by a user.

In some aspects of the automated multi-module cell editing instrument,the transformation module comprises a flow-through electroporationdevice, where the flow-through electroporation device comprises an inletand inlet channel for introduction of the cell sample and assemblednucleic acids into the flow-through electroporation device; an outletand outlet channel for exit of the electroporated cell sample from theflow-through electroporation device; a flow channel intersecting andpositioned between the inlet channel and outlet channel; and two or moreelectrodes, where the two or more electrodes are positioned in the flowchannel between the intersection of the flow channel with the firstinlet channel and the intersection of the flow channel with the outletchannel, in fluid communication with the cell sample in the flowchannel, and configured to apply an electric pulse or electric pulses tothe cell sample. In specific aspects, the flow through electroporationdevice can comprise two or more flow channels in parallel.

Systems for using the automated multi-module cell editing instrument toimplement genomic editing operations within cells are also provided.These systems may optionally include one or more interfaces between theinstrument and other devices or receptacles for cell preparation,nucleic acid preparation, selection of edited cell populations,functional analysis of edited cell populations, storage of edited cellpopulations, and the like.

In addition, methods for using the automated multi-module cell editinginstrument are provided. In some methods, electrocompetent cells areprovided directly to the instrument, preferably at a desired opticaldensity, and transferred to a transformation module. In some methods,cells are transferred to a growth module, where they are grown to adesired optical density. The cells are then transferred from the growthvial to a filtration module where they are concentrated and optionallyrendered electrocompetent. The cells are then transferred to atransformation module.

In some aspects, assembled nucleic acid cassettes are provided directlyto the instrument, and transferred to a transformation module. In someaspects, nucleic acids, such as a vector backbone and one or moreoligonucleotide editing cassettes are transferred to a nucleic acidassembly module either simultaneously or sequentially with the cellintroduction or preparation. In this aspect, nucleic acids areassembled, de-salted (e.g., through a liquid exchange or osmosis), andtransferred to the transformation module to be electroporated into theelectrocompetent cells. Electroporation or transfection takes place inthe transformation module, then the cells are transferred to arecovery/editing module that optionally includes selection of the cellscontaining the one or more genomic edits. Afterrecovery/editing/selection, the cells may be retrieved and used directlyfor research or stored for further research, or another round (ormultiple rounds) of genomic editing can be performed by repeating theediting steps within the instrument.

Also provided are cell libraries created using an automated multi-modulecell editing instrument for nuclease-directed genome editing, where theinstrument comprises: a housing; a receptacle configured to receivecells and one or more rationally designed nucleic acids comprisingsequences to facilitate nuclease-directed genome editing events in thecells; a transformation module for introduction of the nucleic acid(s)into the cells; an editing module for allowing the nuclease-directedgenome editing events to occur in the cells, and a processor configuredto operate the automated multi-module cell editing instrument based onuser input, wherein the nuclease-directed genome editing events createdby the automated instrument result in a cell library comprisingindividual cells with rationally designed edits.

In some aspects, the cell library comprises a saturation mutagenesiscell library. In some aspects, the cell library comprises a promoterswap cell library. In other aspects, the cell library comprises aterminator swap cell library. In yet other aspects, the cell librarycomprises a single nucleotide polymorphism (SNP) swap cell library. Inyet other aspects, the cell library comprises a promoter swap celllibrary.

In some implementations, the library comprises at least 100,000 editedcells, and in yet other implementations, the library comprises at least1,000,000 edited cells.

In some implementations, the nuclease-directed genome editing isRGN-directed genome editing. In a preferred aspect, the instrument isconfigured for the use of an inducible nuclease. The nuclease may be,e.g., chemically induced, virally induced, light induced, temperatureinduced, or heat induced.

In some implementations, the instrument provides multiplexed genomeediting of multiple cells in a single cycle. In some aspects, theinstrument has the ability to edit the genome of at least 5 cells in asingle cycle. In other aspects, the instrument has the ability to editthe genome of at least 100 cells in a single cycle. In yet otheraspects, the instrument has the ability to edit the genome of at least1000 cells in a single cycle. In still other aspects, the instrument hasthe ability to edit the genome of at least 10,000 cells in a singlecycle. In specific aspects, the automated multi-module cell editinginstruments have the ability to edit the genome of at least 10⁴, 10⁵,10⁶ 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴ or more cells in asingle cycle.

The number of genomic sites in a cell population that can be targetedfor editing in a single cycle can be between 2-10,000,000.

In some embodiments that involve recursive editing, the automatedmulti-module cell editing instrument provides introducing two or moregenome edits into cells, with a single genome edit added to the genomesof the cell population for each cycle. Accordingly, some aspects theautomated multi-module cell editing instruments of the presentdisclosure are useful for sequentially providing two or more edits percell in a cell population per cycle, three or more edits per cell in acell population, five or more edits per cell in a population, or 10 ormore edits per cell in a single cycle for a cell population.

In specific embodiments, the automated multi-module cell editinginstrument is able to provide an editing efficiency of at least 10% ofthe cells introduced to the editing module per cycle, preferably anediting efficiency of at least 20% of the cells introduced to theediting module per cycle, more preferably an editing efficiency of atleast 25% of the cells introduced to the editing module per cycle, stillmore preferably an editing efficiency of at least 30% of the cellsintroduced to the editing module automated multi-module cell editinginstrument per cycle, yet more preferably an editing efficiency of atleast 40% of the cells introduced to the editing module per cycle andeven more preferably 50%, 60%, 70%, 80%, 90% or more of the cellsintroduced to the editing module per cycle.

Other features, advantages, and aspects will be described below in moredetail.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of the specification, illustrate one or more embodiments and,together with the description, explain these embodiments. Theaccompanying drawings have not necessarily been drawn to scale. Anyvalues dimensions illustrated in the accompanying graphs and figures arefor illustration purposes only and may or may not represent actual orpreferred values or dimensions. Where applicable, some or all featuresmay not be illustrated to assist in the description of underlyingfeatures. In the drawings:

FIGS. 1A and 1B depict plan and perspective views of an exampleembodiment of an automated multi-module cell processing instrument forthe multiplexed genome editing of multiple cells using a replaceablecartridge(s) as a part of the instrument.

FIGS. 2A and 2B depict side and front views of the automatedmulti-module cell processing instrument of FIGS. 1A and 1B.

FIGS. 2C and 2D depict a second example chassis of an automatedmulti-module cell processing instrument.

FIGS. 3A-3C depict side, cut-away and perspective views of an examplecell wash and/or concentration module for use in an automatedmulti-module cell processing instrument.

FIG. 4 depicts an example combination nucleic acid assembly module andpurification module for use in an automated multi-module cell processinginstrument.

FIG. 5A depicts an example inline electroporation module for use in anautomated multi-module cell processing instrument.

FIGS. 5B and 5C depict an example disposable flow-throughelectroporation module for use in an automated multi-module cellprocessing instrument.

FIGS. 6A-6B depict an example wash cartridge for use in an automatedmulti-module cell processing instrument.

FIGS. 6C-6E depict an example reagent cartridge for use in an automatedmulti-module cell processing instrument.

FIGS. 7A-7C provide a functional block diagram and two perspective viewsof an example filtration module for use in an automated multi-modulecell processing instrument.

FIG. 7D is a perspective views of an example filter cartridge for use inan automated multi-module cell processing instrument.

FIGS. 8A-8F depict example cell growth modules for use in an automatedmulti-module cell processing instrument.

FIG. 9 is a flow chart of an example method for automated multi-modulecell processing.

FIG. 10A is a flow diagram of a first example workflow for automatedprocessing of bacterial cells by an automated multi-module cellprocessing instrument.

FIG. 10B is a flow diagram of a second example workflow for automatedprocessing of a bacterial cells by an automated multi-module cellprocessing instrument.

FIG. 10C is a flow diagram of an example workflow for automated cellprocessing of yeast cells by an automated multi-module cell processinginstrument.

FIG. 11 illustrates an example graphical user interface for providinginstructions to and receiving feedback from an automated multi-modulecell processing instrument.

FIG. 12A is a functional block system diagram of another exampleembodiment of an automated multi-module cell processing instrument forthe multiplexed genome editing of multiple cells.

FIG. 12B is a functional block system diagram of yet another exampleembodiment of an automated multi-module cell processing instrument forthe recursive, multiplexed genome editing of multiple cells.

FIG. 13 is an example control system for use in an automated multi-modecell processing instrument.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The description set forth below in connection with the appended drawingsis intended to be a description of various, illustrative embodiments ofthe disclosed subject matter. Specific features and functionalities aredescribed in connection with each illustrative embodiment; however, itwill be apparent to those skilled in the art that the disclosedembodiments may be practiced without each of those specific features andfunctionalities.

The practice of the techniques described herein may employ thetechniques set forth in Green, et al., Eds. (1999), Genome Analysis: ALaboratory Manual Series (Vols. I-IV); Weiner, Gabriel, Stephens, Eds.(2007), Genetic Variation: A Laboratory Manual; Dieffenbach, Dveksler,Eds. (2003), PCR Primer: A Laboratory Manual; Bowtell and Sambrook(2003), Bioinformatics: Sequence and Genome Analysis; Sambrook andRussell (2006), Condensed Protocols from Molecular Cloning: A LaboratoryManual; and Green and Sambrook, (Molecular Cloning: A Laboratory Manual.4th, ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,2014); Stryer, L. (1995) Biochemistry (4th Ed.) W.H. Freeman, New YorkN.Y.; Gait, “Oligonucleotide Synthesis: A Practical Approach” 1984, IRLPress, London; Nelson and Cox (2000), Lehninger, Principles ofBiochemistry 3^(rd) Ed., W. H. Freeman Pub., New York, N.Y.; and Berg etal. (2002) Biochemistry, 5^(th) Ed., W.H. Freeman Pub., New York, N.Y.,all of which are herein incorporated in their entirety by reference forall purposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “an oligo” refers toone or more oligos that serve the same function, to “the methods”includes reference to equivalent steps and methods known to thoseskilled in the art, and so forth. That is, unless expressly specifiedotherwise, as used herein the words “a,” “an,” “the” carry the meaningof “one or more.” Additionally, it is to be understood that terms suchas “left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,”“length,” “width,” “upper,” “lower,” “interior,” “exterior,” “inner,”“outer” that may be used herein merely describe points of reference anddo not necessarily limit embodiments of the present disclosure to anyparticular orientation or configuration. Furthermore, terms such as“first,” “second,” “third,” etc., merely identify one of a number ofportions, components, steps, operations, functions, and/or points ofreference as disclosed herein, and likewise do not necessarily limitembodiments of the present disclosure to any particular configuration ororientation.

Furthermore, the terms “approximately,” “proximate,” “minor,” andsimilar terms generally refer to ranges that include the identifiedvalue within a margin of 20%, 10% or preferably 5% in certainembodiments, and any values therebetween.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs.

All publications (including patents, published applications, andnon-patent literature) mentioned herein are incorporated by referencefor all purposes, including but not limited to the purpose of describingand disclosing devices, systems, and methods that may be used ormodified in connection with the presently described methods, modules,instruments, and systems.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the disclosure. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges, andare also encompassed within the disclosure, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either both of those includedlimits are also included in the disclosure.

Reference throughout the specification to “one embodiment” or “anembodiment” means that a particular feature, structure, orcharacteristic described in connection with an embodiment is included inat least one embodiment of the subject matter disclosed. Thus, theappearance of the phrases “in one embodiment” or “in an embodiment” invarious places throughout the specification is not necessarily referringto the same embodiment.

Further, the particular features, structures or characteristics may becombined in any suitable manner in one or more embodiments. Further, itis intended that embodiments of the disclosed subject matter covermodifications and variations thereof.

Introduction and Overview

In selected embodiments, the automated multi-module cell editinginstruments, systems and methods described herein can be used inmultiplexed genome editing in living cells, as well as in methods forconstructing libraries of edited cell populations. The automatedmulti-module cell editing instruments disclosed herein can be used witha variety of genome editing techniques, and in particular withnuclease-directed genome editing. The automated multi-module cellediting instruments of the disclosure provide novel methods forintroducing nucleic acid sequences targeting genomic sites for editingthe genome of living cells, including methods for constructing librariescomprising various classes of genomic edits to coding regions,non-coding regions, or both. The automated multi-module cell editinginstruments are particularly suited to introduction of genome edits tomultiple cells in a single cycle, thereby generating libraries of cellshaving one or more genome edits in an automated, multiplexed fashion.The automated multi-module cell editing instruments are also suited tointroduce two or more edits, e.g., edits to different target genomicsites in individual cells of a cell population. Whether one or many,these genome edits are preferably rationally-designed edits; that is,nucleic acids that are designed and created to introduce specific editsto target regions within a cell's genome. The sequences used tofacilitate genome-editing events include sequences that assist inguiding nuclease cleavage, the introduction of a genome edit to a regionof interest, and/or both. These sequences may also include an edit to aregion of the cell's genome to allow the specific rationally designededit in the cell's genome to be tracked. Such methods of introducingedits into cells are taught, e.g., in U.S. Pat. No. 9,982,278, entitled“CRISPR enabled multiplexed genome engineering,” by Gill et al., andU.S. Pat. No. 10,017,760, application Ser. No. 15/632,222, entitled“Methods for generating barcoded combinatorial libraries,” to Gill etal.

Such nucleic acids and oligonucleotides (or “oligos”) are intended toinclude, but are not limited to, a polymeric form of nucleotides thatmay have various lengths, including either deoxyribonucleotides orribonucleotides, or analogs thereof. The nucleic acids andoligonucleotides for use in the illustrative embodiments can be modifiedat one or more positions to enhance stability introduced during chemicalsynthesis or subsequent enzymatic modification or polymerase copying.These modifications include, but are not limited to, the inclusion ofone or more alkylated nucleic acids, locked nucleic acids (LNAs),peptide nucleic acids (PNAs), phosphonates, phosphothioates in theoligomer. Examples of modified nucleotides include, but are not limitedto 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Nucleic acid molecules may also be modified atthe base moiety, sugar moiety or phosphate backbone.

Nuclease-Directed Genome Editing

In selected embodiments, the automated multi-module cell editinginstruments described herein utilize a nuclease-directed genome editingsystem. Multiple different nuclease-based systems exist for providingedits into an organism's genome, and each can be used in either singleediting systems, sequential editing systems (e.g., using differentnuclease-directed systems sequentially to provide two or more genomeedits in a cell) and/or recursive editing systems, (e.g. utilizing asingle nuclease-directed system to introduce two or more genome edits ina cell). Exemplary nuclease-directed genome editing systems aredescribed herein, although a person of skill in the art would recognizeupon reading the present disclosure that other enzyme-directed editingsystems are also useful in the automated multi-module cell editinginstruments of the illustrative embodiments.

It should be noted that the automated systems as set forth herein canuse the nucleases for cleavage of the genome and introduction of an editinto a target genomic region using an instrument of the disclosure.

In particular aspects of the illustrative embodiments, the nucleaseediting system is an inducible system that allows control of the timingof the editing. The inducible system may include inducible expression ofthe nuclease, inducible expression of the editing nucleic acids, orboth. The ability to modulate nuclease activity can reduce off-targetcleavage and facilitate precise genome engineering. Numerous differentinducible systems can be used with the automated multi-module cellediting instruments of the disclosure, as will be apparent to oneskilled in the art upon reading the present disclosure.

In certain aspects, cleavage by a nuclease can be also be used with theautomated multi-module cell editing instruments of the illustrativeembodiments to select cells with a genomic edit at a target region. Forexample, cells that have been subjected to a genomic edit that removes aparticular nuclease recognition site (e.g., via homologousrecombination) can be selected using the automated multi-module cellediting instruments and systems of the illustrative embodiments byexposing the cells to the nuclease following such edit. The DNA in thecells without the genome edit will be cleaved and subsequently will havelimited growth and/or perish, whereas the cells that received the genomeedit removing the nuclease recognition site will not be affected by thesubsequent exposure to the nuclease.

If the cell or population of cells includes a nucleic acid-guidednuclease encoding DNA that is induced by an inducer molecule, thenuclease will be expressed only in the presence of the inducer molecule.Alternatively, if the cell or population of cells includes a nucleicacid-guided nuclease encoding DNA that is repressed by a repressormolecule, the nuclease will be expressed only in the absence of therepressor molecule.

For example, inducible systems for editing using RNA-guided nucleasehave been described, which use chemical induction to limit the temporalexposure of the cells to the RNA-guided nuclease. (US Patent ApplicationPublication 2015/0291966 A1 to Zhang et al., entitled “Inducible DNABinding Proteins and Genome Perturbation Tools and ApplicationsThereof,” filed Jan. 23, 2015; see also inducible lentiviral expressionvectors available at Horizon/Dharmacon, Lafayette, Colo. For additionaltechniques, see e.g., Campbell, Targeting protein function: theexpanding toolkit for conditional disruption, Biochem J., 473(17):2573-2589 (2016).

In other examples, a virus-inducible nuclease can be used to induce geneediting in cells. See, e.g., Dong, Establishment of a highly efficientvirus-inducible CRISPR/Cas9 system in insect cells, Antiviral Res.,130:50-7 (2016). In another example, for inducible expression of nucleicacid directed nucleases, variants can be switched on and off inmammalian cells with 4-hydroxytamoxifen (4-HT) by fusing the nucleasewith the hormone-binding domain of the estrogen receptor (ERT2). (Liu,et al., Nature Chemical Biology, 12:980-987 (2016) and see InternationalPatent Application Publication WO 2017/078631 A1 to Tan, entitled“Chemical-Inducible Genome Engineering Technology,” filed Nov. 7, 2016.

In addition, a number of gene regulation control systems have beendeveloped for the controlled expression of genes in cells, bothprokaryotic and eukaryotic. These systems include thetetracycline-controlled transcriptional activation system(Tet-On/Tet-Off, Clontech, Inc. (Palo Alto, Calif.), the Lac SwitchInducible system (U.S. Pat. No. 4,833,080 to Brent et al., entitled“Regulation of eucaryotic gene expression”), the ecdysone-inducible geneexpression system (No et al., Ecdysone-inducible gene expression inmammalian cells and transgenic mice, PNAS, 93(8):3346-3351 (1996)), andthe cumate gene-switch system (Mullick, et al., The cumate gene-switch:a system for regulated expression in mammalian cells, BMC Biotechnology,6:43 (2006)).

The cells that can be edited using the automated multi-module cellediting instruments of the illustrative embodiments include anyprokaryotic, archaeal or eukaryotic cell. For example, prokaryotic cellsfor use with the present illustrative embodiments can be gram positivebacterial cells, e.g., Bacillus subtilis, or gram negative bacterialcells, e.g., E. coli cells. Eukaryotic cells for use with the automatedmulti-module cell editing instruments of the illustrative embodimentsinclude any plant cells and any animal cells, e.g. fungal cells, insectcells, amphibian cells nematode cells, or mammalian cells.

Zinc-finger Nuclease Genome Editing

In selected embodiments, the automated multi-module cell editinginstruments described herein perform zinc-finger nuclease genomeediting. Zinc-finger nucleases (ZFNs) are artificial restriction enzymesgenerated by fusing a zinc finger DNA-binding domain to a DNA-cleavagedomain. Zinc finger domains can be engineered to target-specific regionsin an organism's genome. (Urnov et al., Nature Reviews Genetics,11:636-646 (2010); International Patent Application Publication WO2003/087341 A2 to Carroll et al., entitled “Targeted ChromosomalMutagenesis Using Zinc Finger Nucleases,” filed Jan. 22, 2003). Usingthe endogenous DNA repair machinery of an organism, ZFNs can be used toprecisely alter a target region of the genome. ZFNs can be used todisable dominant mutations in heterozygous individuals by producingdouble-strand breaks (“DSBs”) in the DNA in the mutant allele, whichwill, in the absence of a homologous template, be repaired bynon-homologous end-joining (NHEJ). NHEJ repairs DSBs by joining the twoends together and usually produces no mutations, provided that the cutis clean and uncomplicated. (Durai et al., Zinc finger nucleases:custom-designed molecular scissors for genome engineering of plant andmammalian cells, Nucleic Acids Res., 33(18):5978-90 (2005)). This repairmechanism can be used to induce errors in the genome via indels orchromosomal rearrangement, often rendering the gene products coded atthat location non-functional.

Alternatively, DNA can be introduced into a genome in the presence ofexogenous double-stranded DNA fragments using homology dependent repair(HDR). The dependency of HDR on a homologous sequence to repair DSBs canbe exploited by inserting a desired sequence within a sequence that ishomologous to the flanking sequences of a DSB which, when used as atemplate by HDR system, leads to the creation of the desired changewithin the genomic region of interest.

Multiple pairs of ZFNs can also be used to completely remove entirelarge segments of genomic sequence (Lee et al., Genome Res., 20 (1):81-9 (2009); and US Patent Application Publication 2011/0082093 A1 toGregory et al. entitled “Methods and Compositions for TreatingTrinucleotide Repeat Disorders,” filed Jul. 28, 2010). Expanded CAG/CTGrepeat tracts are the genetic basis for more than a dozen inheritedneurological disorders including Huntington's disease, myotonicdystrophy, and several spinocerebellar ataxias. It has been demonstratedin human cells that ZFNs can direct DSBs to CAG repeats and shrink therepeat from long pathological lengths to short, less toxic lengths(Mittelman, et al., Zinc-finger directed double-strand breaks within CAGrepeat tracts promote repeat instability in human cells, PNAS USA, 106(24): 9607-12 (2009); and US Patent Application Publication 2013/0253040A1 to Miller et al. entitled “Methods and Compositions for TreatingHuntington's Disease,” filed Feb. 28, 2013).

Meganuclease Genome Editing

In selected embodiments, the automated multi-module cell editing,modules instruments and systems described herein perform meganucleasegenome editing. Meganucleases were identified in the 1990s, andsubsequent work has shown that they are particularly promising tools forgenome editing, as they are able to efficiently induce homologousrecombination, generate mutations in coding or non-coding regions of thegenome, and alter reading frames of the coding regions of genomes. (See,e.g., Epinat, et al., A novel engineered meganuclease induces homologousrecombination in eukaryotic cells, e.g., yeast and mammalian cells,Nucleic Acids Research, 31(11): 2952-2962; and U.S. Pat. No. 8,921,332to Choulika et al. entitled “Chromosomal Modification Involving theInduction of Double-stranded DNA Cleavage and Homologous Recombinationat the Cleavage Site,” issued Dec. 30, 2014.) The high specificity ofmeganucleases gives them a high degree of precision and much lower celltoxicity than other naturally occurring restriction enzymes.

Transcription Activator-like Effector Nuclease Editing

In selected embodiments, the automated multi-module cell editingmodules, instruments and systems described herein perform transcriptionactivator-like effector nuclease editing. Transcription activator-likeeffector nucleases (TALENs) are restriction enzymes that can beengineered to cut specific sequences of DNA. They are made by fusing aTAL effector DNA-binding domain to a DNA cleavage domain (a nucleasewhich cuts DNA strands). Transcription activator-like effector nucleases(TALENs) can be engineered to bind to practically any desired DNAsequence, so when combined with a nuclease, DNA can be cut at specificlocations. (See, e.g., Miller, et al., A TALE nuclease architecture forefficient genome editing, Nature Biotechnology, 29 (2): 143-8 (2011);Boch, Nature Biotech.,TALEs of genome targeting, 29(2): 135-6 (2011);International Patent Application Publication WO 2010/079430 A1 to Bonaset al. entitled “Modular DNA-binding Domains and Methods of Use,” filedJan. 12, 2010; International Patent Application Publication WO2011/072246 A2 to Voytas et al. entitled “TAL Effector-Mediated DNAModification,” filed Dec. 10, 2010).

Like ZFNs, TALENs can edit genomes by inducing DSBs. The TALEN-createdsite-specific DSBs at target regions are repaired through NHEJ or HDR,resulting in targeted genome edits. TALENs can be used to introduceindels, rearrangements, or to introduce DNA into a genome through NHEJin the presence of exogenous double-stranded DNA fragments.

RNA-Guided Nuclease (RGN) Editing

In certain aspects, the genome editing of the automated multi-modulecell editing instruments of the illustrative embodiments utilizeclustered regularly interspaced short palindromic repeats (CRISPR)techniques, in which RNA-guided nucleases (RGNs) are used to editspecific target regions in an organism's genome. By delivering the RGNcomplexed with a synthetic guide RNA (gRNA) into a cell, the cell'sgenome can be cut at a desired location, allowing edits to the targetregion of the genome. The guide RNA helps the RGN proteins recognize andcut the DNA of the target genome region. By manipulating the nucleotidesequence of the guide RNA, the RGN system could be programmed to targetany DNA sequence for cleavage.

The RGN system used with the automated multi-module cell editinginstruments of the illustrative embodiments can perform genome editingusing any RNA-guided nuclease system with the ability to both cut andpaste at a desired target genomic region. In certain aspects, theRNA-guided nuclease system may use two separate RNA molecules as a gRNA,e.g., a CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA).In other aspects, the gRNA may be a single gRNA that includes both thecrRNA and tracrRNA sequences.

In certain aspects, the genome editing both introduces a desired DNAchange to a target region and removes the proto-spacer motif (PAM)region from the target region, thus precluding any additional editing ofthe genome at that target region, e.g., upon exposure to a RNA-guidednuclease complexed with a synthetic gRNA complementary to the targetregion. In this aspect, a first editing event can be, e.g., anRGN-directed editing event or a homologous recombination event, andcells having the desired edit can be selected using an RGN complexedwith a synthetic gRNA complementary to the target region. Cells that didnot undergo the first editing event will be cut, and thus will notcontinue to be viable under appropriate selection criteria. The cellscontaining the desired mutation will not be cut, as they will no longercontain the necessary PAM site, and will continue to grow and propagatein the automated multi-module cell editing instrument.

When the RGN protein system is used for selection, it is primarily thecutting activity that is needed; thus the RNA-guided nuclease proteinsystem can either be the same as used for editing, or may be a RGNprotein system that is efficient in cutting using a particular PAM site,but not necessarily efficient in editing at the site. One importantaspect of the nuclease used for selection is the recognition of the PAMsite that is replaced using the editing approach of the previous genomeediting operation.

Genome Editing by Homologous Recombination

In other aspects, the genome editing of the automated multi-module cellediting instruments of the illustrative embodiments can utilizehomologous recombination methods including the cre-lox technique and theFRET technique. Site-specific homologous recombination differs fromgeneral homologous recombination in that short specific DNA sequences,which are required for the recombinase recognition, are the only sitesat which recombination occurs. Site-specific recombination requiresspecialized recombinases to recognize the sites and catalyze therecombination at these sites. A number of bacteriophage- andyeast-derived site-specific recombination systems, each comprising arecombinase and specific cognate sites, have been shown to work ineukaryotic cells for the purpose of DNA integration and are thereforeapplicable for use in the present invention, and these include thebacteriophage P1 Cre/lox, yeast FLP-FRT system, and the Dre system ofthe tyrosine family of site-specific recombinases. Such systems andmethods of use are described, for example, in U.S. Pat. Nos. 7,422,889;7,112,715; 6,956,146; 6,774,279; 5,677,177; 5,885,836; 5,654,182; and4,959,317, which are incorporated herein by reference to teach methodsof using such recombinases. Other systems of the tyrosine family such asbacteriophage lambda Int integrase, HK2022 integrase, and in additionsystems belonging to the separate serine family of recombinases such asbacteriophage phiC31, R4Tp901 integrases are known to work in mammaliancells using their respective recombination sites, and are alsoapplicable for use in the present invention. Exemplary methodologies forhomologous recombination are described in U.S. Pat. Nos. 6,689,610;6,204,061; 5,631,153; 5,627,059; 5,487,992; and 5,464,764, each of whichis incorporated by reference in its entirety.

Instrument Architecture

FIGS. 1A and 1B depict an example automated multi-module cell processinginstrument 100 utilizing cartridge-based source materials (e.g.,reagents, enzymes, nucleic acids, wash solutions, etc.). The instrument100, for example, may be designed as a desktop instrument for use withina laboratory environment. The instrument 100 may incorporate a mixtureof reusable and disposable elements for performing various stagedoperations in conducting automated genome cleavage and/or editing incells. The cartridge-based source materials, for example, may bepositioned in designated areas on a deck 102 of the instrument 100 foraccess by a robotic handling instrument 108. As illustrated in FIG. 1B,the deck 102 may include a protection sink such that contaminantsspilling, dripping, or overflowing from any of the modules of theinstrument 100 are contained within a lip of the protection sink.

Turning to FIG. 1A, the instrument 100, in some implementations,includes a reagent cartridge 104 for introducing DNA samples and othersource materials to the instrument 100, a wash cartridge 106 forintroducing eluent and other source materials to the instrument 100, anda robot handling system 108 for moving materials between modules (forexample, modules 110 a, 110 b, and 110 c) cartridge receptacles (forexample, receptacles of cartridges 104 and 106), and storage units(e.g., units 112, 114, 116, and 118) of the instrument 100 to performautomated genome cleavage and/or editing. Upon completion of processingof the cell supply 106, in some embodiments, cell output may betransferred by the robot handling instrument 108 to a storage unit orreceptacle placed in, e.g., reagent cartridge 104 or wash cartridge 106for temporary storage and later retrieval.

The robotic handling system 108, for example, may include an airdisplacement pump 120 to transfer liquids from the various materialsources of the cartridges 104, 106 to the various modules 110 and to thestorage unit, which may be a receptacle in reagent cartridge 104 or washcartridge 106. In other embodiments, the robotic handling system 108 mayinclude a pick and place head (not illustrated) to transfer containersof source materials (e.g., tubes or vials) from the reagent cartridge104 and/or the wash cartridge 106 to the various modules 110. In someembodiments, one or more cameras or other optical sensors (not shown)confirm proper movement and position of the robotic handling apparatusalong a gantry 122.

In some embodiments, the robotic handling system 108 uses disposabletransfer tips provided in a transfer tip supply 116 (e.g., pipette tiprack) to transfer source materials, reagents (e.g., nucleic acidassembly), and cells within the instrument 100. Used transfer tips 116,for example, may be discarded in a solid waste unit 112. In someimplementations, the solid waste unit 112 contains a kicker to removetubes, tips, vials, and/or filters from the pick and place head ofrobotic handling system 108. For example, as illustrated the robotichandling system 108 includes a filter pickup head 124.

In some embodiments, the instrument 100 includes electroporator cuvetteswith sippers that connect to the air displacement pump 120. In someimplementations, cells and reagent are aspirated into theelectroporation cuvette through a sipper, and the cuvette is moved toone or more modules 110 of the instrument 100.

In some implementations, the instrument 100 is controlled by aprocessing system 126 such as the processing system 1310 of FIG. 13. Theprocessing system 126 may be configured to operate the instrument 100based on user input. For example, user input may be received by theinstrument 100 through a touch screen control display 128. Theprocessing system 126 may control the timing, duration, temperature andother operations of the various modules 110 of the instrument 100.Turning to FIG. 1B, the processing system 126 may be connected to apower source 150 for the operation of the instrument 100.

Returning to FIG. 1A, the reagent cartridge 104, as illustrated,includes sixteen reservoirs (a matrix of 5×3 reservoirs, plus anadditional reservoir) and a flow-through transformation module(electroporation device) 110 c. The wash cartridge 106 may be configuredto accommodate large tubes or reservoirs to store, for example, washsolutions, or solutions that are used often throughout an iterativeprocess. Further, in some embodiments, the wash cartridge 106 mayinclude a number of smaller tubes, vials, or reservoirs to retainsmaller volumes of, e.g., source media as well as a receptacle orrepository for edited cells. For example, the wash cartridge 106 may beconfigured to remain in place when two or more reagent cartridges 104are sequentially used and replaced. Although the reagent cartridge 104and wash cartridge 106 are shown in FIG. 1A as separate cartridges, inother embodiments, the contents of the wash cartridge 106 may beincorporated into the reagent cartridge 104. In further embodiments,three or more cartridges may be loaded into the automated multi-modulecell processing instrument 100. In certain embodiments, the reagentcartridge 104, wash cartridge 106, and other components of the modules110 in the automated multi-module cell processing instrument 100 arepackaged together in a kit.

The wash and reagent cartridges 104, 106, in some implementations, aredisposable kits provided for use in the automated multi-module cellprocessing instrument 100. For example, the user may open and positioneach of the reagent cartridge 104 and the wash cartridge 106 within achassis of the automated multi-module cell processing instrument priorto activating cell processing. Example chassis are discussed in furtherdetail below in relation to FIGS. 2A through 2D.

Components of the cartridges 104, 106, in some implementations, aremarked with machine-readable indicia, such as bar codes, for recognitionby the robotic handling system 108. For example, the robotic handlingsystem 108 may scan containers within each of the cartridges 104, 106 toconfirm contents. In other implementations, machine-readable indicia maybe marked upon each cartridge 104, 106, and the processing system of theautomated multi-module cell processing instrument 100 may identify astored materials map based upon the machine-readable indicia.

Turning to FIGS. 6A-6B, in some embodiments, the wash cartridge 106 is awash cartridge 600 including a pair of large bottles 602, a set of foursmall tubes 604, and a large tube 606 held in a cartridge body 608. Eachof the bottles 602 and tubes 604, 606, in some embodiments, is sealedwith a pierceable foil for access by an automated liquid handlingsystem, such as a sipper or pipettor. In other embodiments, each of thebottles 602 and tubes 604, 606 includes a sealable access gasket. Thetop of each of the bottles 602 and tubes 604, 606, in some embodiments,is marked with machine-readable indicia (not illustrated) for automatedidentification of the contents.

In some embodiments, the large bottles 602 each contain wash solution.The wash solution may be a same or different wash solutions. In someexamples, wash solutions may contain, e.g., buffer, buffer and 10%glycerol, 80% ethanol.

In some implementations, a cover 610 secures the bottles 602 and tubes604, 606 within the cartridge body 608. Turning to FIG. 6B, the cover610 may include apertures for access to each of the bottles 602 andtubes 604, 606. Further, the cover 610 may include machine-readableindicia 612 for identifying the type of cartridge (e.g., accessing a mapof the cartridge contents). Alternatively, each aperture may be markedseparately with the individual contents.

Turning to FIGS. 6C-E, in some implementations, the reagent cartridge104 is a reagent cartridge 620 including a set of sixteen small tubes orvials 626, and flow-through electroporation module 624, held in acartridge body 622. Each of the small tubes or vials 626, in someembodiments, is sealed with pierceable foil for access by an automatedliquid handling system, such as a sipper or pipettor. In otherembodiments, each of the small tubes or vials 626 includes a sealableaccess gasket. The top of each of the small tubes or vials 626, in someembodiments, is marked with machine-readable indicia (not illustrated)for automated identification of the contents. The machine-readableindicia may include a bar code, QR code, or other machine-readablecoding. Other automated means for identifying a particular container caninclude color coding, symbol recognition (e.g., text, image, icon,etc.), and/or shape recognition (e.g., a relative shape of thecontainer). Rather than being marked upon the vessel itself, in someembodiments, an upper surface of the cartridge body and/or the cartridgecover may contain machine-readable indicia for identifying contents. Thesmall tubes or vials may each be of a same size. Alternatively, multiplevolumes of tubes or vials may be provided in the reagent cartridge 620.In an illustrative example, each tube or vial may be designed to holdbetween 2 and 20 mL, between 4 and 10 mL, or about 5 mL.

In an illustrative example, the small tubes or vials 626 may each holdone the following materials: a vector backbone, oligonucleotides,reagents for isothermal nucleic acid assembly, a user-supplied cellsample, an inducer agent, magnetic beads in buffer, ethanol, anantibiotic for cell selection, reagents for eluting cells and nucleicacids, an oil overlay, other reagents, and cell growth and/or recoverymedia.

In some implementations, a cover 628 secures the small tubes or vials626 within the cartridge body 622. Turning to FIG. 6D, the cover 628 mayinclude apertures for access to each of the small tubes or vials 626.Three large apertures 632 are outlined in a bold (e.g., blue) band toindicate positions to add user-supplied materials. The user-suppliedmaterials, for example, may include a vector backbone, oligonucleotides,and a cell sample. Further, the cover 610 may include machine-readableindicia 630 for identifying the type of cartridge (e.g., accessing a mapof the cartridge contents). Alternatively, each aperture may be markedseparately with the individual contents. In some implementations, toensure positioning of user-supplied materials, the vials or tubesprovided for filling in the lab environment may have unique shapes orsizes such that the cell sample vial or tube only fits in the cellsample aperture, the oligonucleotides vial or tube only fits in theoligonucleotides aperture, and so on.

Turning back to FIG. 1A, also illustrated is the robotic handling system108 including the gantry 122. In some examples, the robotic handlingsystem 108 may include an automated liquid handling system such as thosemanufactured by Tecan Group Ltd. of Mannedorf, Switzerland, HamiltonCompany of Reno, Nev. (see, e.g., WO2018015544A1 to Ott, entitled“Pipetting device, fluid processing system and method for operating afluid processing system”), or Beckman Coulter, Inc. of Fort Collins,Colo. (see, e.g., US20160018427A1 to Striebl et al., entitled “Methodsand systems for tube inspection and liquid level detection”). Therobotic handling system 108 may include an air displacement pipettor120. The reagent cartridges 104, 106 allow for particularly easyintegration with the liquid handling instrumentation of the robotichandling system 108 such as air displacement pipettor 120. In someembodiments, only the air displacement pipettor 120 is moved by thegantry 122 and the various modules 110 and cartridges 104, 106 remainstationary. Pipette tips 116 may be provided for use with the airdisplacement pipettor 120.

In some embodiments, an automated mechanical motion system (actuator)(not shown) additionally supplies XY axis motion control or XYZ axismotion control to one or more modules 110 and/or cartridges 104, 106 ofthe automated multi-module cell processing system 100. Used pipette tips116, for example, may be placed by the robotic handling system in awaste repository 112. For example, an active module may be raised tocome into contact-accessible positioning with the robotic handlingsystem or, conversely, lowered after use to avoid impact with therobotic handling system as the robotic handling system is movingmaterials to other modules 110 within the automated multi-module cellprocessing instrument 100.

The automated multi-module cell processing instrument 100, in someimplementations, includes the flow-through electroporation module 110 cincluded in the reagent cartridge 104. A flow-through electroporationconnection bridge 132, for example, is engaged with the flow-throughelectroporation device after the cells and nucleic acids are transferredinto the device via an input channel. The bridge 132 provides both aliquid-tight seal and an electrical connection to the electrodes, aswell as control for conducting electroporation within theelectroporation module 110 c. For example, the electroporationconnection bridge 132 may be connected to flow-through electroporationcontrols 134 within an electronics rack 136 of the automatedmulti-module cell processing instrument 100.

In some implementations, the automated multi-module cell processinginstrument 100 includes dual cell growth modules 110 a, 110 b. The cellgrowth modules 110 a, 110 b, as illustrated each include a rotating cellgrowth vial 130 a, 130 b. At least one of the cell growth modules 110 a,110 b may additionally include an integrated filtration module (notillustrated). In alternative embodiments, a filtration module or a cellwash and concentration module may instead be separate from cell growthmodules 110 a, 110 b (e.g., as described in relation to cell growthmodule 1210 a and filtration module 1210 b of FIGS. 12A and 12B). Thecell growth modules 110 a, 110 b, for example, may each include thefeatures and functionalities discussed in relation to the cell growthmodule 800 of FIGS. 8A-F.

A filtration portion of one or both of the cell growth modules 110 a,110 b, in some embodiments, use replaceable filters stored in a filtercassette 118. For example, the robotic handling system may include thefilter pick-up head 124 to pick up and engage filters for use with oneor both of the cell growth modules 110 a, 110 b. The filter pick-up headtransfers a filter to the growth module, pipettes up the cells from thegrowth module, then washes and renders the cells electrocompetent. Themedium from the cells, and the wash fluids are disposed in waste module114.

In some implementations, automated multi-module cell processinginstrument 100 includes a nucleic acid assembly and purificationfunction (e.g., nucleic acid assembly module) for combining materialsprovided in the reagent cartridge 104 into an assembled nucleic acid forcell editing. Further, a desalting or purification operation purifiesthe assembled nucleic acids and de-salts the buffer such that thenucleic acids are more efficiently electroporated into the cells. Thenucleic acid assembly and purification feature may include a reactionchamber or tube receptacle (not shown) and a magnet (not shown).

Although the example instrument 100 is illustrated as including aparticular arrangement of modules 110, this implementation is forillustrative purposes only. For example, in other embodiments, more orfewer modules 110 may be included within the instrument 100, anddifferent modules may be included such as, e.g., a module for cellfusion to produce hybridomas and/or a module for protein production.Further, certain modules may be replicated within certain embodiments,such as the duplicate cell growth modules 110 a, 110 b of FIG. 1A.

In some embodiments, the cells are modified prior to introduction ontothe automated multi-module cell editing instrument. For example, thecells may be modified by using a λ red system to replace a target genewith an antibiotic resistance gene, usually for kanamycin orchloramphenicol. (See Datsenko and Wanner, One-step inactivation ofchromosomal genes in Escherichia coli K-12 using PCR products, PNAS USA,97(12):6640-5 (2000); U.S. Pat. No. 6,509,156 B1 to Stewart et al.entitled “DNA Cloning Method Relying on the E. coli recE/recTRecombination System,” issued Jan. 21, 2003.) In some embodiments, thecells may have already been transformed or transfected with a vectorcomprising an expression cassette for a nuclease. In another example, adesired gene edit may be introduced to the cell population prior tointroduction to the automated multi-module cell editing instrument(e.g., using homology directed repair), and the system used to selectthese edits using a nuclease and/or add additional edits to the cellpopulation.

FIGS. 2A through 2D illustrate example chassis 200 and 230 for use indesktop versions of an automated multi-module cell processinginstrument. For example, the chassis 200 and 230 may have a width ofabout 24-48 inches, a height of about 24-48 inches and a depth of about24-48 inches. Each of the chassis 200 and 230 may be designed to holdmultiple modules and disposable supplies used in automated cellprocessing. Further, each chassis 200 and 250 may mount a robotichandling system for moving materials between modules.

FIGS. 2A and 2B depict a first example chassis 200 of an automatedmulti-module cell processing instrument. As illustrated, the chassis 200includes a cover 202 having a handle 204 and hinges 206 for lifting thecover 202 and accessing an interior of the chassis 200. A cooling grate214 may allow for air flow via an internal fan (not shown). Further, thechassis 200 is lifted by adjustable feet 220. The feet 220, for example,may provide additional air flow beneath the chassis 200. A controlbutton 216, in some embodiments, allows for single-button automatedstart and stop of cell processing within the chassis 200.

Inside the chassis 200, in some implementations, a robotic handlingsystem 208 is disposed along a gantry 210 above materials cartridges 212a, 212 b and modules. Control circuitry, liquid handling tubes, air pumpcontrols, valves, thermal units (e.g., heating and cooling units) andother control mechanisms, in some embodiments, are disposed below a deckof the chassis 200, in a control box region 218.

Although not illustrated, in some embodiments, a display screen may bepositioned upon a front face of the chassis 200, for example covering aportion of the cover 202. The display screen may provide information tothe user regarding a processing status of the automated multi-modulecell processing instrument. In another example, the display screen mayaccept inputs from the user for conducting the cell processing.

FIGS. 2C and 2D depict a second example chassis 230 of an automatedmulti-module cell processing instrument. The chassis 230, asillustrated, includes a transparent door 232 with a hinge 234. Forexample, the door may swing to the left of the page to provide access toa work area of the chassis. The user, for example, may open thetransparent door 232 to load supplies, such as reagent cartridges andwash cartridges, into the chassis 230.

In some embodiments, a front face of the chassis 230 further includes adisplay (e.g., touch screen display device) 236 illustrated to the rightof the door 232. The display 236 may provide information to the userregarding a processing status of the automated multi-module cellprocessing instrument. In another example, the display 236 may acceptinputs from the user for conducting the cell processing.

An air grate 238 on a right face of the chassis 230 may provide for airflow within a work area (e.g., above the deck) of the chassis 230 (e.g.,above a deck). A second air grate 240 on a left of the chassis 230 mayprovide for air flow within a control box region 242 (e.g., below thedeck) of the chassis 230. Although not illustrated, in some embodiments,feet such as the feet 220 of the chassis 200 may raise the chassis 230above a work surface, providing for further air flow.

Inside the chassis 230, in some implementations, a robotic handlingsystem 248 is disposed along a gantry 250 above cartridges 252 a, 252 b,material supplies 254 a, 254 b (e.g., pipette tips and filters), andmodules 256 (e.g., dual growth vials). Control circuitry, liquidhandling tubes, air pump controls, valves, and other control mechanisms,in some embodiments, are disposed below a deck of the chassis 230, inthe control box region 242.

In some embodiments, a liquid waste unit 246 is mounted to the leftexterior wall of the chassis 230. The liquid waste unit 246, forexample, may be mounted externally to the chassis 230 to avoid potentialcontamination and to ensure prompt emptying and replacement of theliquid waste unit 246.

Nucleic Acid Assembly Module

Certain embodiments of the automated multi-module cell editinginstruments of the present disclosure include a nucleic acid assemblymodule within the instrument. The nucleic acid assembly module isconfigured to accept the nucleic acids necessary to facilitate thedesired genome editing events. The nucleic acid assembly module may alsobe configured to accept the appropriate vector backbone for vectorassembly and subsequent transformation into the cells of interest.

In general, the term “vector” refers to a nucleic acid molecule capableof transporting another nucleic acid to which it has been linked.Vectors include, but are not limited to, nucleic acid molecules that aresingle-stranded, double-stranded, or partially double-stranded; nucleicacid molecules that include one or more free ends, no free ends (e.g.circular); nucleic acid molecules that include DNA, RNA, or both; andother varieties of polynucleotides known in the art. One type of vectoris a “plasmid,” which refers to a circular double stranded DNA loop intowhich additional DNA segments can be inserted, such as by standardmolecular cloning techniques. Another type of vector is a viral vector,where virally-derived DNA or RNA sequences are present in the vector forpackaging into a virus (e.g. retroviruses, replication defectiveretroviruses, adenoviruses, replication defective adenoviruses, andadeno-associated viruses). Viral vectors also include polynucleotidescarried by a virus for transfection into a host cell. Certain vectorsare capable of autonomous replication in a host cell into which they areintroduced (e.g. bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively-linked.Such vectors are referred to herein as “expression vectors.” Commonexpression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. Further discussion of vectors is provided herein.

Recombinant expression vectors can include a nucleic acid in a formsuitable for transformation, and for some nucleic acids sequences,translation and expression of the nucleic acid in a host cell, whichmeans that the recombinant expression vectors include one or moreregulatory elements—which may be selected on the basis of the host cellsto be used for expression—that are operatively-linked to the nucleicacid sequence to be expressed. Within a recombinant expression vector,“operably linked” is intended to mean that the nucleotide sequence ofinterest is linked to the regulatory element(s) in a manner that allowsfor transcription, and for some nucleic acid sequences, translation andexpression of the nucleotide sequence (e.g. in an in vitrotranscription/translation system or in a host cell when the vector isintroduced into the host cell). Appropriate recombination and cloningmethods are disclosed in U.S. patent application Ser. No. 10/815,730,entitled “Recombinational Cloning Using Nucleic Acids HavingRecombination Sites” published Sep. 2, 2004 as US 2004-0171156 A1, thecontents of which are herein incorporated by reference in their entiretyfor all purposes.

In some embodiments, a regulatory element is operably linked to one ormore elements of a targetable nuclease system so as to drivetranscription, and for some nucleic acid sequences, translation andexpression of the one or more components of the targetable nucleasesystem.

In some embodiments, a vector may include a regulatory element operablylinked to a polynucleotide sequence encoding a nucleic acid-guidednuclease. The polynucleotide sequence encoding the nucleic acid-guidednuclease can be codon optimized for expression in particular cells, suchas prokaryotic or eukaryotic cells. Eukaryotic cells can be yeast,fungi, algae, plant, animal, or human cells. Eukaryotic cells may bethose of or derived from a particular organism, such as a mammal,including but not limited to human, mouse, rat, rabbit, dog, ornon-human mammal including non-human primate. In addition oralternatively, a vector may include a regulatory element operably likedto a polynucleotide sequence, which, when transcribed, forms a guideRNA.

The nucleic acid assembly module can be configured to perform a widevariety of different nucleic acid assembly techniques in an automatedfashion. Nucleic acid assembly techniques that can be performed in thenucleic acid assembly module of the disclosed automated multi-modulecell editing instruments include, but are not limited to, those assemblymethods that use restriction endonucleases, including PCR, BioBrickassembly (U.S. Pat. No. 9,361,427 to Hillson entitled “Scar-lessMulti-part DNA Assembly Design,” issued Jun. 7, 2016), Type IIS cloning(e.g., GoldenGate assembly; European Patent Application Publication EP 2395 087 A1 to Weber et al. entitled “System and Method of ModularCloning,” filed Jul. 6, 2010), and Ligase Cycling Reaction (de Kok S,Rapid and Reliable DNA Assembly via Ligase Cycling Reaction, ACS SynthBiol., 3(2):97-106 (2014); Engler, et al., PLoS One, A One Pot, OneStep, Precision Cloning Method with High Throughput Capability,3(11):e3647 (2008); U.S. Pat. No. 6,143,527 to Pachuk et al. entitled“Chain Reaction Cloning Using a Bridging Oligonucleotide and DNALigase,” issued Nov. 7, 2000). In other embodiments, the nucleic acidassembly techniques performed by the disclosed automated multi-modulecell editing instruments are based on overlaps between adjacent parts ofthe nucleic acids, such as Gibson Assembly®, CPEC, SLIC, Ligase Cyclingetc. Additional assembly methods include gap repair in yeast (Bessa,Improved gap repair cloning in yeast: treatment of the gapped vectorwith Taq DNA polymerase avoids vector self-ligation, Yeast,29(10):419-23 (2012)), gateway cloning (Ohtsuka, Lantibiotics: mode ofaction, biosynthesis and bioengineering, Curr Pharm Biotechnol,10(2):244-51 (2009); U.S. Pat. No. 5,888,732 to Hartley et al., entitled“Recombinational Cloning Using Engineered Recombination Sites,” issuedMar. 30, 1999; U.S. Pat. No. 6,277,608 to Hartley et al. entitled“Recominational Cloning Using Nucleic Acids Having Recombination Sites,”issued Aug. 21, 2001), and topoisomerase-mediated cloning (Udo, AnAlternative Method to Facilitate cDNA Cloning for Expression Studies inMammalian Cells by Introducing Positive Blue White Selection in VacciniaTopoisomerase I-Mediated Recombination, PLoS One, 10(9):e0139349 (2015);U.S. Pat. No. 6,916,632 B2 to Chestnut et al. entitled “Methods andReagents for Molecular Cloning,” issued Jul. 12, 2005). These and othernucleic acid assembly techniques are described, e.g., in Sands andBrent, Overview of Post Cohen-Boyer Methods for Single Segment Cloningand for Multisegment DNA Assembly, Curr Protoc Mol Biol.,113:3.26.1-3.26.20 (2016); Casini et al., Bricks and blueprints: methodsand standards for DNA assembly, Nat Rev Mol Cell Biol., (9):568-76(2015); Patron, DNA assembly for plant biology: techniques and tools,Curr Opinion Plant Biol., 19:14-9 (2014)).

The nucleic acid assembly is temperature controlled depending upon thetype of nucleic acid assembly used in the automated multi-module cellediting instrument. For example, when PCR is utilized in the nucleicacid assembly module, the module will have a thermocycling capabilityallowing the temperatures to cycle between denaturation, annealing andextension. When single temperature assembly methods are utilized in thenucleic acid assembly module, the module will have the ability to reachand hold at the temperature that optimizes the specific assembly processbeing performed. These temperatures and the duration for maintainingthese temperatures can be determined by a preprogrammed set ofparameters executed by a script, or manually controlled by the userusing the processing system of the automated multi-module cellprocessing instrument.

In one embodiment, the nucleic acid assembly module is a module toperform assembly using a single, isothermal reaction, such as thatillustrated in FIG. 4. The isothermal assembly module is configured toperform the molecular cloning method using the single, isothermalreaction. Certain isothermal assembly methods can combine simultaneouslyup to 15 nucleic acid fragments based on sequence identity. The assemblymethod provides, in some embodiments, nucleic acids to be assembledwhich include an approximate 20-40 base overlap with adjacent nucleicacid fragments. The fragments are mixed with a cocktail of threeenzymes—an exonuclease, a polymerase, and a ligase-along with buffercomponents. Because the process is isothermal and can be performed in a1-step or 2-step method using a single reaction vessel, isothermalassembly reactions are ideal for use in an automated multi-module cellprocessing instrument. The 1-step method allows for the assembly of upto five different fragments using a single step isothermal process. Thefragments and the master mix of enzymes are combined and incubated at50° C. for up to one hour. For the creation of more complex constructswith up to fifteen fragments or for incorporating fragments from 100 bpup to 10 kb, typically the 2-step is used, where the 2-step reactionrequires two separate additions of master mix; one for the exonucleaseand annealing step and a second for the polymerase and ligation steps.

FIG. 4 illustrates an example isothermal nucleic acid assembly module400 with integrated purification. The isothermal nucleic acid assemblymodule 400 includes a chamber 402 having an access gasket 404 fortransferring liquids to and from the isothermal nucleic acid assemblymodule 400 (e.g., via a pipette or sipper). In some embodiments, theaccess gasket 404 is connected to a replaceable vial which is positionedwithin the chamber 402. For example, a user or robotic manipulationsystem may place the vial within the isothermal nucleic acid assemblymodule 400 for processing.

The chamber 402 shares a housing 406 with a resistive heater 408. Once asample has been introduced to the chamber 402 of the isothermal nucleicacid assembly module 400, the resistive heater 408 may be used to heatthe contents of the chamber 402 to a desired temperature. Thermalramping may be set based upon the contents of the chamber 402 (e.g., thematerials supplied through the access gasket 404 via pipettor or sipperunit of the robotic manipulation system). The processing system of theautomated multi-module cell processing system may determine the targettemperature and thermal ramping plan. The thermal ramping and targettemperature may be controlled through monitoring a thermal sensor suchas a thermistor 410 included within the housing 406. In a particularembodiment, the resistive heater 408 is designed to maintain atemperature within the housing 406 of between 20° and 80° C., between25° and 75° C., between 37° and 65° C., between 40° and 60° C., between45° and 55° C. or preferably about 50° C.

Purification Module

In some embodiments, when a nucleic acid assembly module is included inthe automated multi-module cell editing instrument, the instrument alsocan include a purification module to remove unwanted components of thenucleic acid assembly mixture (e.g., salts, minerals) and, in certainembodiments, concentrate the assembled nucleic acids. Examples ofmethods for exchanging the liquid following nucleic acid assemblyinclude magnetic beads (e.g., SPRI or Dynal (Dynabeads) by InvitrogenCorp. of Carlsbad, Calif.), silica beads, silica spin columns, glassbeads, precipitation (e.g., using ethanol or isopropanol), alkalinelysis, osmotic purification, extraction with butanol, membrane-basedseparation techniques, filtration etc.

In one aspect, the purification module provides filtration, e.g.,ultrafiltration. For example, a range of microconcentrators fitted withanisotropic, hydrophilic-generated cellulose membranes of varyingporosities is available. (See, e.g., Millipore SCX microconcentratorsused in Juan, Li-Jung, et al. “Histone deacetylases specificallydown-regulate p53-dependent gene activation.” Journal of BiologicalChemistry 275.27 (2000): 20436-20443.). In another example, thepurification and concentration involves contacting a liquid sampleincluding the assembled nucleic acids and an ionic salt with an ionexchanger including an insoluble phosphate salt, removing the liquid,and eluting the nucleic acid from the ion exchanger.

In a specific aspect of the purification module, SPRI beads can be usedwhere 0.6-2.0× volumes of SPRI beads can be added to the nucleic acidassembly. The nucleic acid assembly product becomes bound to the SPRIbeads, and the SPRI beads are pelleted by automatically positioning amagnet close to the tube, vessel, or chamber harboring the pellet. Forexample, 0.6-2.0× volumes of SPRI beads can be added to the nucleic acidassembly. The SPRI beads, for example, may be washed with ethanol, andthe bound nucleic acid assembly product is eluted, e.g., in water, Trisbuffer, or 10% glycerol.

In a specific aspect, a magnet is coupled to a linear actuator thatpositions the magnet. In some implementations, the nucleic acid assemblymodule is a combination assembly and purification module designed forintegrated assembly and purification. For example, as discussed above inrelation to an isothermal nucleic acid assembly module, once sufficienttime has elapsed for the isothermal nucleic acid assembly reaction totake place, the contents of the chamber 402 (e.g., the isothermalnucleic acid assembly reagents and nucleic acids), in some embodiments,are combined with magnetic beads (not shown) to activate thepurification process. The SPRI beads in buffer are delivered to thecontents of the isothermal nucleic acid assembly module, for example, bya robotic handling system. Thereafter, a solenoid 412, in someembodiments, is actuated by a magnet to excite the magnetic beadscontained within the chamber 402, The solenoid, in a particular example,may impart between a 2 pound magnetic pull force and a 5 pound pullforce, or approximately a 4 pound magnetic pull force to the magneticbeads within the chamber 402. The contents of the chamber 402 may beincubated for sufficient time for the assembled vector andoligonucleotides to bind to the magnetic beads.

After binding, in some implementations, the bound isothermal nucleicacid. assembly mix (e.g., isothermal nucleic acid assemblyreagents+assembled vector and oligonucleotides) is removed from theisothermal nucleic acid assembly module and the nucleic acids attachedto the beads are washed one to several times with 80% ethanol. Oncewashed, the nucleic acids attached to the beads are eluted into bufferand are transferred to the transformation module.

In some implementations, a vial is locked in position in the chamber 402for processing. For example, a user may press the vial beyond a detectin the chamber 402 designed to retain the vial upon engagement with apipettor or sipper. In another example, the user may twist the vial intoposition, thus engaging a protrusion to a corresponding channel andbarring upward movement. A position sensor (not illustrated) may ensureretraction of the vial. The position sensor, in a particular embodiment,is a magnetic sensor detecting engagement between a portion of thechamber 402 and the vial. In other embodiments, the position sensor isan optical sensor detecting presence of the vial at a retractedposition. In embodiments using a channel and protrusion, a mechanicswitch pressed down by the protrusion may detect engagement of the vial.

Growth Module

As the nucleic acids are being assembled, the cells may be grown inpreparation for editing. The cell growth can be monitored by opticaldensity (e.g., at OD 600 nm) that is measured in a growth module, and afeedback loop is used to adjust the cell growth so as to reach a targetOD at a target time. Other measures of cell density and physiologicalstate that can be measured include but are not limited to, pH, dissolvedoxygen, released enzymes, acoustic properties, and electricalproperties.

In some aspects, the growth module includes a culture tube in a shakeror vortexer that is interrogated by a spectrophotometer or fluorimeter.The shaker or vortexer can heat or cool the cells and cell growth ismonitored by real-time absorbance or fluorescence measurements. In oneaspect, the cells are grown at 25° C.-40° C. to an OD600 absorbance of1-10 ODs. The cells may also be grown at temperature ranges from 25°C.-35° C., 25° C.-30° C., 30° C.-40° C., 30° C.-35° C., 35° C.-40° C.,40° C.-50° C., 40° C.-45° C. or 44° C.-50° C. In another aspect, thecells are induced by heating at 42° C.-50° C. or by adding an inducingagent. The cells may also be induced by heating at ranges from 42°C.-46° C., 42° C.-44° C., 44° C.-46° C., 44° C.-48° C., 46° C.-48° C.,46° C.-50° C., or 48° C.-50° C. In some aspects, the cells are cooled to0° C.-10° C. after induction. The cells may also be cooled totemperature ranges of 0° C.-5° C., 0° C.-2° C., 2° C.-4° C., 4° C.-6°C., 6° C.-8° C., 8° C.-10° C., or 5° C.-10° C. after induction.

FIG. 8A shows one embodiment of a rotating growth vial 800 for use witha cell growth device, such as cell growth device 850 illustrated inFIGS. 8B-C. The rotating growth vial 800, in some implementations, is atransparent container having an open end 804 for receiving liquid mediaand cells, a central vial region 806 that defines the primary containerfor growing cells, a tapered-to-constricted region 818 defining at leastone light path 808, 810, a closed end 816, and a drive engagementmechanism 812. The rotating growth vial 800 may have a centrallongitudinal axis 820 around which the vial 800 rotates, and the lightpaths 808, 810 may be generally perpendicular to the longitudinal axisof the vial. In some examples, first light path 810 may be positioned inthe lower constricted portion of the tapered-to-constricted region 818.The drive engagement mechanism 812, in some implementations, engageswith a drive mechanism (e.g., actuator, motor (not shown)) to rotate thevial 800. The actuator may include a drive shaft 874 for a drive motor864 (FIG. 8D).

In some embodiments, the rotating growth vial 800 includes a secondlight path 808, for example, in the upper tapered region of thetapered-to-constricted region 818. In some examples, the walls definingthe upper tapered region of the tapered-to-constricted region 818 forthe second light path 808 may be disposed at a wider angle relative tothe longitudinal axis 820 than the walls defining the lower constrictedportion of the tapered-to-constricted region 810 for the first lightpath 810. Both light paths 808, 810, for example, may be positioned in aregion of the rotating growth vial 800 that is constantly filled withthe cell culture (cells+growth media), and is not affected by therotational speed of the growth vial 800. As illustrated, the secondlight path 808 is shorter than the first light path 810 allowing forsensitive measurement of optical density (OD) values when the OD valuesof the cell culture in the vial are at a high level (e.g., later in thecell growth process), whereas the first light path 810 allows forsensitive measurement of OD values when the OD values of the cellculture in the vial are at a lower level (e.g., earlier in the cellgrowth process).

The rotating growth vial 800 may be reusable, or preferably, therotating growth vial is consumable. In some embodiments, the rotatinggrowth vial 800 is consumable and can be presented to the userpre-filled with growth medium, where the vial 800 is sealed at the openend 804 with a foil seal. A medium-filled rotating growth vial packagedin such a manner may be part of a kit for use with a stand-alone cellgrowth device or with a cell growth module that is part of an automatedmulti-module cell processing system. To introduce cells into the vial, auser need only pipette up a desired volume of cells and use the pipettetip to punch through the foil seal of the vial 800. Alternatively, ofcourse, an automated instrument may transfer cells from, e.g., a reagentcartridge, to the growth vial. The growth medium may be provided in thegrowth vial or may also be transferred from a reagent cartridge to thegrowth vial before the addition of cells. Open end 804 may include anextended lip 802 to overlap and engage with the cell growth device 850(FIGS. 8B-C). In automated instruments, the rotating growth vial 800 maybe tagged with a barcode or other identifying means that can be read bya scanner or camera that is part of the processing system 1310 asillustrated in FIG. 13.

In some implementations, the volume of the rotating growth vial 800 andthe volume of the cell culture (including growth medium) may varygreatly, but the volume of the rotating growth vial 800 should be largeenough for the cell culture in the growth vial 800 to get properaeration while the vial 800 is rotating. In practice, the volume of therotating growth vial 800 may range from 1-250 ml, 2-100 ml, from 5-80ml, 10-50 ml, or from 12-35 ml. Likewise, the volume of the cell culture(cells+growth media) should be appropriate to allow proper aeration inthe rotating growth vial 800. Thus, the volume of the cell cultureshould be approximately 10-85% of the volume of the growth vial 800, or15-80% of the volume of the growth vial, or 20-70%, 30-60%, or 40-50% ofthe volume of the growth vial. In one example, for a 35 ml growth vial800, the volume of the cell culture would be from about 4 ml to about 27ml.

The rotating growth vial 800, in some embodiments, is fabricated from abio-compatible transparent material-or at least the portion of the vial800 including the light path(s) is transparent. Additionally, materialfrom which the rotating growth vial 800 is fabricated should be able tobe cooled to about 0° C. or lower and heated to about 75° C. or higher,such as about 2° C. or to about 70° C., about 4° C. or to about 60° C.,or about 4° C. or to about 55° C. to accommodate both temperature-basedcell assays and long-term storage at low temperatures. Further, thematerial that is used to fabricate the vial is preferably able towithstand temperatures up to 55° C. without deformation while spinning.Suitable materials include glass, polyvinyl chloride, polyethylene,polyamide, polyethylene, polypropylene, polycarbonate, poly(methylmethacrylate) (PMMA), polysulfone, polyurethane, and co-polymers ofthese and other polymers. Preferred materials include polypropylene,polycarbonate, or polystyrene. In some embodiments, the rotating growthvial 800 is inexpensively fabricated by, e.g., injection molding orextrusion.

FIG. 8B illustrates a top view of a rotating growth vial 800 b, which isan alternative implementation of the rotating growth vial 800. In someexamples, the vial 800 b may include one or more paddles 822 affixed toan inner surface that protrude toward the center of the vial 800 b. Thevial 800 b shown in FIG. 8B includes three paddles 822 that aresubstantially equally spaced around the periphery of the vial 800 b, butin other examples, the vial 800 b may include two, four, or more paddles822. The paddles, in some implementations, provide high mixing andaeration within the vial 800 b rotating within a cell growth device,which facilitates microbial growth.

FIGS. 8C-D illustrate views of an example cell growth device 850 thatreceives the rotating growth vial 800. In some embodiments, the cellgrowth device 850 rotates to heat or cool the cells or cell growthwithin the vial 800 to a predetermined temperature range. In someimplementations, the rotating growth vial 800 can be positioned inside amain housing 852 with the extended lip 802 of the vial 800 extendingpast an upper surface of the main housing 852. In some aspects, theextended lip 802 provides a grasping surface for a user inserting orwithdrawing the vial 800 from the main housing 852 of the device 850.Additionally, when fully inserted into the main housing 852, a lowersurface of the extended lip 802 abuts an upper surface of the mainhousing 852. In some examples, the main housing 852 of the cell growthdevice 850 is sized such that outer surfaces of the rotating growth vial800 abut inner surfaces of the main housing 852 thereby securing thevial 800 within the main housing 852. In some implementations, the cellgrowth device 850 can include end housings 854 disposed on each side ofthe main housing 854 and a lower housing 856 disposed at a lower end ofthe main housing 852. In some examples, the lower housing 856 mayinclude flanges 858 that can be used to attach the cell growth device850 to a temperature control (e.g, heating/cooling) mechanism or otherstructure such as a chassis of an automated cell processing system.

As shown in FIG. 8D, the cell growth device 850, in someimplementations, can include an upper bearing 860 and lower bearing 862positioned in main housing 852 that support the vertical load of arotating growth vial 800 that has been inserted into the main housing852. In some examples, the cell growth device 850 may also include aprimary optical port 866 and a secondary optical port 868 that arealigned with the first light path 810 and second light path 808 of thevial 800 when inserted into the main housing 852. In some examples, theprimary and secondary optical ports 866, 868 are gaps, openings, orportions of the main housing constructed from transparent materials thatallow light to pass through the vial 800 to perform cell growth ODmeasurements. In addition to the optical ports 866, 868, the cell growthdevice 850 may include an emission board 870 that provides one or moreillumination sources for the light path(s), and detector board 872 todetect the light after the light travels through the cell culture liquidin the rotating growth vial 800. In one example, the illuminationsources disposed on the emission board 870 may include light emissiondiodes (LEDs) or photodiodes that provide illumination at one or moretarget wavelengths commensurate with the growth media typically used incell culture (whether, e.g., mammalian cells, bacterial cells, animalcells, yeast cells).

In some implementations, the emission board 870 and/or detector board872 are communicatively coupled through a wired or wireless connectionto a processing system (e.g., processing system 126, 1220, 1310) thatcontrols the wavelength of light output by the emission board 870 andreceives and processes the illumination sensed at the detector board872. The remotely controllable emission board 870 and detector board872, in some aspects, provide for conducting automated OD measurementsduring the course of cell growth. For example, the processing system126, 1220 may control the periodicity with which OD measurements areperformed, which may be at predetermined intervals or in response to auser request Further, the processing system 126, 1220 can use the sensordata received from the detector board 872 to perform real-time ODmeasurements and adjust cell growth conditions (e.g., temperature,speed/direction of rotation).

In some embodiments, the lower housing 856 may contain drive motor 864that generates rotational motion that causes the rotating growth vial800 to spin within the cell growth device 850. In some implementations,the motor 864 may include a drive shaft 874 that engages a lower end ofthe rotating growth vial 800. The motor 864 that generates rotationalmotion for the rotating growth vial 800, in some embodiments, is abrushless DC type drive motor with built-in drive controls that can beconfigured to maintain a constant revolution per minute (RPM) between 0and about 3000 RPM. Alternatively, other motor types such as a stepper,servo, or brushed DC motors can be used. Optionally, the motor 864 mayalso have direction control to allow reversing of the rotationaldirection, and a tachometer to sense and report actual RPM. In otherexamples, the motor 864 can generate oscillating motion by reversing thedirection of rotation at a predetermined frequency. In one example, thevial 800 is rotated in each direction for one second at a speed of 350RPM. The motor 864, in some implementations, is communicatively coupledthrough a wired or wireless communication network to a processing system(e.g., processing system 126, 1220) that is configured to control theoperation of the motor 864, which can include executing protocolsprogrammed into the processor and/or provided by user input, for exampleas described in relation to module controller 1330 of FIG. 13. Forexample, and the motor 864 can be configured to vary the speed and/orrotational direction of the vial 800 to cause axial precession of thecell culture thereby enhancing mixing in order to prevent cellaggregation and increase aeration. In some examples, the speed ordirection of rotation of the motor 864 may be varied based on opticaldensity sensor data received from the detector board 872.

In some embodiments, main housing 852, end housings 854 and lowerhousing 856 of the cell growth device 856 may be fabricated from arobust material including aluminum, stainless steel, and other thermallyconductive materials, including plastics. These structures or portionsthereof can be created through various techniques, e.g., metalfabrication, injection molding, creation of structural layers that arefused, etc. While in some examples the rotating growth vial 800 isreusable, in other embodiments, the vial 800 is preferably isconsumable. The other components of the cell growth device 850, in someaspects, are preferably reusable and can function as a stand-alonebenchtop device or as a module in an automated multi-module cellprocessing system.

In some implementations, the processing system that is communicativelycoupled to the cell growth module may be programmed with information tobe used as a “blank” or control for the growing cell culture. A “blank”or control, in some examples, is a vessel containing cell growth mediumonly, which yields 100% transmittance and 0 OD, while the cell samplesdeflect light rays and will have a lower percentage transmittance andhigher OD. As the cells grow in the media and become denser,transmittance decreases and OD increases. The processor of the cellgrowth module, in some implementations, may be programmed to usewavelength values for blanks commensurate with the growth mediatypically used in cell culture (whether, e.g., mammalian cells,bacterial cells, animal cells, yeast cells). Alternatively, a secondspectrophotometer and vessel may be included in the cell growth module,where the second spectrophotometer is used to read a blank at designatedintervals.

FIG. 8E illustrates another type of cell growth device 880 that usesshaking, rather than rotation, to control temperature and promote mixingand aeration within a cell growth vial 890 (FIG. 8F). The cell growthdevice 880, in some examples, is smaller in size than conventional benchtop shakers for integration into automated multi-module cell processingsystems. In some implementations, the cell growth device 880 includes ahousing 884 that receives cell growth vial 890. The cell growth device880 can, in some examples, include a motor assembly positioned beneaththe vial 890 that generates an orbital motion of the vial 890 based onthe speed of the motor. In one example, the vial 890 travels in an orbitin a horizontal plane at 600 to 900 RPM, such as at 750 RPM, which issignificantly faster than larger bench top shakers that orbit at around250 RPM. In some aspects, the shaking motion is generated in at leastone horizontal plane. In some examples, the cell growth vial 890 usedwith the shaking cell growth device 880 is a conical bottom tubesubstantially similar in shape to a flask that is used in a conventionalbench shaker. Similar to the rotating cell growth device 850, the cellgrowth device 880 may include illumination board 870 and detector board872 for taking automated OD measurements over the course of cell growth.In some examples, a light source 882 may be coupled to the cell growthdevice 880 that generates the illumination that is measured by adetector board, which in some examples, is located beneath the vial 890or on an opposite side of the vial 890 from the light source 882.

To reduce background of cells that have not received a genome edit, thegrowth module may also allow a selection process to enrich for theedited cells. For example, the introduced nucleic acid can include agene, which confers antibiotic resistance or another selectable marker.Alternating the introduction of selectable markers for sequential roundsof editing can also eliminate the background of unedited cells and allowmultiple cycles of the automated multi-module cell editing instrument toselect for cells having sequential genome edits.

Suitable antibiotic resistance genes include, but are not limited to,genes such as ampicillin-resistance gene, tetracycline-resistance gene,kanamycin-resistance gene, neomycin-resistance gene,canavanine-resistance gene, blasticidin-resistance gene,hygromycin-resistance gene, puromycin-resi stance gene, andchloramphenicol-resistance gene. In some embodiments, removing dead cellbackground is aided using lytic enhancers such as detergents, osmoticstress, temperature, enzymes, proteases, bacteriophage, reducing agents,or chaotropes. In other embodiments, cell removal and/or media exchangeis used to reduce dead cell background.

Cell Wash and/or Concentration Module

The cell wash and/or concentration module can utilize any method forexchanging the liquids in the cell environment, and may concentrate thecells or allow them to remain in essentially the same or greater volumeof liquid as used in the nucleic acid assembly module. Further, in someaspects, the processes performed in the cell wash module also render thecells electrocompetent, by, e.g., use of glycerol in the wash.

Numerous different methods can be used to wash the cells, includingdensity gradient purification, dialysis, ion exchange columns,filtration, centrifugation, dilution, and the use of beads forpurification.

In some aspects, the cell wash and/or concentration module utilizes acentrifugation device. In other aspects, the cell wash and/orconcentration module utilizes a filtration module. In yet other aspects,beads are coupled to moieties that bind to the cell surface. Thesemoieties include but are not limited to antibodies, lectins, wheat germagglutinin, mutated lysozymes, and ligands.

In other aspects, the cells are engineered to be magnetized, allowingmagnets to pellet the cells after wash steps. Mechanism of cellmagnetization can include but not limited to ferritin proteinexpression.

The cell wash and/or concentration module, in some implementations, is acentrifuge assembly module. Turning to FIGS. 3A-C, in someimplementations, a centrifuge assembly module 300 includes a top door302 designed for actuation by a robotic handling system (not shown) todeliver nucleic acid assembly materials (e.g., oligos, vector backbone,enzymes, etc.) to one or more vials 304 a, b situated in vial buckets306 a, b connected to a rotor 308. In some embodiments, the robotichandling system delivers the vials 304 a,b to the centrifuge assemblymodule 300. In other embodiments, a user disposes the vials 304 a, bwithin the vial buckets 306 a, b. The vial buckets 306 a, b in someembodiments, are connected to the rotor 308 via a hinged connection suchthat the via buckets 306 a,b may swing outwards during rotation. Inother embodiments, the position of the buckets 306 a,b is fixed.

The centrifuge assembly module 300, in some embodiments, is climaticallycontrolled. For example, the internal temperature may be managed bycooling coils 310 and insulation 312. Coolant supply and return lines314 may pump coolant to the cooling coils 310, thereby cooling a chamber316 of the centrifuge assemble module 300. In some examples, thecentrifuge assembly module 300 may be designed to cool the chamber 316to between 0° and 10° C., between 2° and 8° C., and most preferably toabout 4° C. Further, condensation control may be provided to limithumidity within the chamber 316. Climatic control, in some embodiments,is set through a processing system of the automated multi-module cellprocessing instrument. For example, the processing system may directsignals to interfaces of circuitry 320.

In some embodiments, a motor 318 rotationally drives the rotor 308.Acceleration and deceleration of the motor 318 and thus the rotor 308may be controlled by a processing system of the automated multi-modulecell processing instrument. As illustrated, a motion sensor 322 (e.g.,accelerometer or gyroscope) is positioned at a base of the motor 318 tomonitor rotational parameters. Alternatively, a motion sensor (notillustrated) such as an accelerometer or gyroscope may be placed withinthe chamber 316 to monitor rotational parameters. The processing system,for example, may monitor signals from the motion sensor and analyzeconditions to enact a safety shutdown if rotation is outside parameters.In an illustrative embodiment, the rotor arm may be designed to rotateat up to 10000 revolutions per minute (RPM), up to 8000 RPM, or up toabout 6500 RPM. The processing system may modify the rotational speedbased upon materials supplied to the centrifuge assembly module 300.

The cell wash and/or concentration module, in some implementations, is afiltration module. Turning to FIG. 7A, a block diagram illustratesexample functional units of a filtration module 700. In someimplementations, a main control 702 of the filtration module 700includes a first liquid pump 704 a to intake wash fluid 706 and a secondliquid pump 704 b to remove liquid waste to a liquid waste unit 708(e.g., such as the liquid waste unit 114 of FIG. 1A or liquid waste unit1228 of FIGS. 12A and 12B). A flow sensor 712 may be disposed on aconnector 714 to the liquid waste unit 708 to monitor release of liquidwaste from the filtration module. A valve 716 (a three-way valve asillustrated) may be disposed on a connector 718 to the wash fluid 716 toselectively connect the wash fluid 716 and the filtration module 700.

The filtration module 700, in some implementations, includes a filtermanifold 720 for filtering and concentrating a cell sample. The filtermanifold 720 may include one or more temperature sensor(s) 722 andpressure sensor (s) 724 to monitor flow and temperature of the washfluid and/or liquid waste. The sensors 722, 724, in some embodiments,are monitored and analyzed by a processing system of the automatedmulti-mode cell processing system, such as the processing system 1310 ofFIG. 13. The filter manifold 720 may include one or more valves 726 fordirecting flow of the wash fluid and/or liquid waste. The processingsystem of the automated multi-mode cell processing instrument, forexample, may actuate the valves according to a set of instructions fordirecting filtration by the filtration module 700.

The filtration module 700 includes at least one filter 730. Examples offilters suitable for use in the filtration module 700 include membranefilters, ceramic filters and metal filters. The filter may be used inany shape; the filter may for example be cylindrical or essentiallyflat. The filter selected for a given operation or a given workflow, insome embodiments, depends upon the type of workflow (e.g., bacterial,yeast, viral, etc.) or the volumes of materials being processed. Forexample, while flat filters are relatively low cost and commonly used,filters with a greater surface area, such as cylindrical filters, mayaccept higher flow rates. In another example, hollow filters maydemonstrate lower recovery rates when processing small volumes of sample(e.g., less than about 10 ml). For example, for use with bacteria, itmay be preferable that the filter used is a membrane filter,particularly a hollow fiber filter. With the term “hollow fiber” ismeant a tubular membrane. The internal diameter of the tube, in someexamples, is at least 0.1 mm, more preferably at least 0.5 mm, mostpreferably at least 0.75 mm and preferably the internal diameter of thetube is at most 10 mm, more preferably at most 6 mm, most preferably atmost 1 mm. Filter modules having hollow fibers are commerciallyavailable from various companies, including G.E. Life Sciences(Marlborough, Mass.) and InnovaPrep (Drexel, Mo.) (see, e.g.,US20110061474A1 to Page et al., entitled “Liquid to Liquid BiologicalParticle Concentrator with Disposable Fluid Path”).

In some implementations, the filtration module 700 includes a filterejection means 728 (e.g., actuator) to eject a filter 730 post use. Forexample, a user or the robotic handling system may push the filter 730into position for use such that the filter is retained by the filtermanifold 720 during filtration. After filtration, to remove the usedfilter 730, the filter ejection actuator 728 may eject the filter 730,releasing the filter 730 such that the user or the robotic handlingsystem may remove the used filter 730 from the filtration module 700.The used filter 730, in some examples, may be disposed within the solidwaste unit 112 of FIGS. 1A-1B, solid waste unit 1218 of FIGS. 12A and12B, or returned to a filter cartridge 740, as illustrated in FIG. 7D.

Turning to FIG. 7D, in some implementations, filters 730 provided in thefilter cartridge 740 disposed within the chassis of the automatedmulti-module cell processing instrument are transported to thefiltration module 700 by a robotic handling system (e.g., the robotichandling system 108 described in relation to FIGS. 1A and 1B, or robotichandling system 1218 of FIGS. 12A and 12B) and positioned within thefiltration module 700 prior to use.

The filtration module 700, in some implementations, requires periodiccleaning. For example, the processing system may alert a user whencleaning is required through the user interface of the automatedmulti-module cell processing instrument and/or through a wirelessmessaging means (e.g., text message, email, and/or personal computingdevice application). A decontamination filter, for example, may beloaded into the filtration module 700 and the filtration module 700 maybe cleaned with a wash solution and/or alcohol mixture.

In some implementations, the filtration module 700 is in fluidconnection with a wash cartridge 710 (such as the wash cartridge 600 ofFIG. 6A) containing the wash fluid 716 via the connector 718. Forexample, upon positioning by the user of the wash cartridge 710 withinthe chassis of the automated multi-module cell processing instrument,the connector 718 may mate with a bottom inlet of the wash cartridge710, creating a liquid passage between the wash fluid 716 and thefiltration module 700.

Turning to FIGS. 7B and 7C, in some implementations, a dual filterfiltration module 750 includes dual filters 752, 754 disposed over dualwash reservoirs 754. In an example, each filter may be a hollow corefiber filter having 0.45 um pores and greater than 85 cm² area. The washreservoirs 754, in some examples, may be 50 mL, 100 mL, or over 200 mLin volume. In some embodiments, the wash reservoirs 754 are disposed ina wash cartridge 756, such as the wash or reagent cartridge 600 of FIG.6A.

The processing system of the automated multi-module cell processinginstrument, in some implementations, controls actuation of the dualfilters 752 in an X (horizontal) and Z (vertical) direction to positionthe filters 752 a, 752 b in the wash reservoirs 754. In a particularexample, the dual filters 752 may be move in concert along the X axisbut have independent Z axis range of motion.

As illustrated, the dual filters 752 of the filtration module 750 areconnected to a slender arm body 758. In some embodiments, any pumps andvalves of the filtration module 750 may be disposed remotely from thebody 758 (e.g., within a floor of the chassis of the automatedmulti-module cell processing instrument). In this manner, the filtrationmodule 750 may replace much bulkier conventional commercial filtrationmodules.

Further, in some embodiments, the filtration module 750 is in liquidcommunication with a waste purge system designed to release liquid wasteinto a liquid waste storage unit, such as the storage unit 708 of FIG.7A or the liquid waste storage unit 114 of FIG. 1A or 1228 of FIGS. 12Aand 12B.

Transformation Module

The transformation module may implement any cell transformation ortransfection techniques routinely used by those of skill in the arts oftransfection, transformation and microfluidics. Transformation can takeplace in microfuge tubes, test tubes, cuvettes, multi-well plates,microfibers, and flow instruments. Temperature and control of thetransformation module can be controlled using a processing system suchas the processing system 1310 of FIG. 13, with controls set by the userand/or through a script provided to the processing system.

Transformation is intended to include to a variety of art-recognizedtechniques for introducing an exogenous nucleic acid sequence (e.g.,DNA) into a target cell, and the term “transformation” as used hereinincludes all transformation and transfection techniques. Such methodsinclude, but are not limited to, electroporation, lipofection,optoporation, injection, microprecipitation, microinjection, liposomes,particle bombardment, sonoporation, laser-induced poration, beadtransfection, calcium phosphate or calcium chloride co-precipitation, orDEAE-dextran-mediated transfection. Cells can also be prepared forvector uptake using, e.g., a sucrose or glycerol wash. Additionally,hybrid techniques that exploit the capabilities of mechanical andchemical transfection methods can be used, e.g., magnetofection, atransfection methodology that combines chemical transfection withmechanical methods. In another example, cationic lipids may be deployedin combination with gene guns or electroporators. Suitable materials andmethods for transforming or transfecting target cells can be found,e.g., in Green and Sambrook, Molecular Cloning: A Laboratory Manual,4th, ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,2014).

The medium or buffer used to suspend the cells and material (reagent) tobe electroporated into the cells for the electroporation process may bea medium or buffer including, but not limited to, MEM, DMEM, IMDM, RPMI,Hanks', PBS or Ringer's solution, where the media may be provided in thereagent cartridge as part of a kit. For electroporation of mosteukaryotic cells, the medium or buffer usually contains salts tomaintain a proper osmotic pressure. The salts in the medium or bufferalso render the medium conductive. For electroporation of very smallprokaryotic cells such as bacteria, sometimes water or 10% glycerol isused as a low conductance medium to allow a very high electric fieldstrength. In that case, the charged molecules to be delivered stillrender water-based medium more conductive than the lipid-based cellmembranes and the medium may still be roughly considered as conductiveparticularly in comparison to cell membranes.

The compound to be electroporated into the cells of choice can be anycompound known in the art to be useful for electroporation, such asnucleic acids, oligonucleotides, polynucleotides, DNA, RNA, peptides,proteins and small molecules like hormones, cytokines, chemokines,drugs, or drug precursors.

It is important to use voltage sufficient for achieving electroporationof material into the cells, but not too much voltage as too much powerwill decrease cell viability. For example, to electroporate a suspensionof a human cell line, 200 volts is needed for a 0.2 ml sample in a 4mm-gap cuvette with exponential discharge from a capacitor of about 1000μF. However, if the same 0.2 ml cell suspension is placed in a longercontainer with 2 cm electrode distance (5 times of cuvette gapdistance), the voltage required would be 1000 volts, but a capacitor ofonly 40 μF ( 1/25 of 1000 μF) is needed because the electric energy froma capacitor follows the equation of:E=0.5U²Cwhere E is electric energy, U is voltage and C is capacitance. Thereforea high voltage pulse generator is actually easy to manufacture becauseit needs a much smaller capacitor to store a similar amount of energy.Similarly, it would not be difficult to generate other wave forms ofhigher voltages.

The electroporation devices of the disclosure can allow for a high rateof cell transformation in a relatively short amount of time. The rate ofcell transformation is dependent on the cell type and the number ofcells being transformed. For example, for E. Coli, the electroporationdevices can provide a cell transformation rate of 1 to 10¹⁰ cells persecond, 10⁴ to 10⁷ per second, 10⁵ to 10⁸ per second, or 10⁶ to 10⁹ persecond. The electroporation devices also allow transformation of batchesof cells ranging from 1 cell to 10¹⁰ cells in a single transformationprocedure using the device.

The efficiency of the transformation using the electroporation devicesof the disclosure can result in at least 10% of the cells beingsufficiently porated to allow delivery of the biological molecule.Preferably, the efficiency of the transformation using theelectroporation devices of the disclosure can result in at least 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 75%, 80%, 85%, 90%, 95% orgreater of the cells being sufficiently porated to allow delivery of thebiological molecule.

In some embodiments, the electroporation is performed in a cuvette, awell, a tube, a chamber, a flow cell, a channel, or a pipette tip. Inother embodiments, the cuvette, well, tube, or chamber is filled andemptied from the bottom. In some embodiments, the cuvette contains asipper connected to the bottom.

FIG. 5A depicts an example single-unit electroporation device 500(electroporation module) including, from top to bottom, a housing 502that encloses an engagement member 504 configured to engage with apipette such as an automatic air displacement pipette (not shown), and afilter 506. In addition to the housing 502, there is an electroporationcuvette 510 portion of the electroporation device 500 includingelectrodes 512, and walls 514 of the electroporation chamber 516. Thechamber, in some examples, may range between 0.01-100 mm in width,1-5,000 mm in height, and 1-20,000 μl in volume; between 0.03-50 mm inwidth, 50-2,000 mm in height, and 500-10,000 μl in volume; or between0.05-30 mm in width, 2-500 mm in height, and 25-4,500 μl in volume.

In some embodiments, a first reservoir 508 may be placed between thefilter 506 and the electroporation chamber 516, the first reservoirbeing in fluid communication with electroporation chamber 516 andproviding an empty repository for any cell sample that may be taken inpast the electroporation chamber 516. The first reservoir 508, in someexamples, may range between 0.1-150 mm in width, 0.1-250 mm in height,and 0.5-10,000 μl in volume; between 0.3-100 mm in width, 30-150 mm inheight, and 20-4,000 μl in volume; or between 0.5-100 mm in width,0.5-100 mm in height, and 5-2,000 μl in volume.

In some implementations, the electroporation device 500 may additionallyinclude another reservoir 524 in fluid communication with the firstreservoir 508 (through filter 506). The second reservoir 524 may beplaced between the filter 506 and the engagement member 504 to protectthe pipette from contamination by any liquids that may make it past thefilter 506. The second reservoir 524, in some examples, may rangebetween 0.1-250 mm in width, 0.2-1000 mm in height, and 0.1-2,500 μl involume; between 0.1-150 mm in width, 50-400 mm in height, and 1-1,000 Win volume; or between 0.2-100 mm in width, 0.5-200 mm in height, and2-600 μl in volume.

In some embodiments, a sipper 518 is in fluid communication with andcoupled to the electroporation chamber 516, the sipper 518 having an endproximal 520 to the electroporation chamber 516 and an end distal 522from the electroporation chamber 516. The distal end 522 of the sipper518 may allow for uptake and dispensing of the cell sample from theelectroporation device 500. The sipper 518, in some embodiments, is partof a robotic manipulation system. The sipper 518, in some examples, maybe made from plastics such as polyvinyl chloride, polyethylene,polyamide, polyethylene, polypropylene, acrylonitrile butadiene,polycarbonate, polyetheretheketone (PEEK), polysulfone and polyurethane,co-polymers of these and other polymers, glass (such as a glasscapillary), and metal tubing such as aluminum, stainless steel, orcopper. Exemplary materials include crystal styrene and cyclic olephinco-polymers. PEEK is a preferred plastic given it is low in price andeasily fabricated. The sipper 518, in some examples, may range between0.02-2,000 mm in width, 0.25-2,000 mm in height, and 1-2,000 μl involume; between 0.02-1,250 mm in width, 250-1,500 mm in height, and1.5-1,500 μl in volume; or between 0.02-10 mm in width, 4.0-1,000 mm inheight, and 2.5-1,000 μl in volume.

The housing 502 and engagement member 504 of the electroporation device500, in some examples, can be made from silicone, resin, polyvinylchloride, polyethylene, polyamide, polyethylene, polypropylene,acrylonitrile butadiene, polycarbonate, polyetheretheketone (PEEK),polysulfone and polyurethane, co-polymers of these and other polymers.Similarly, the walls 512 of the electroporation chamber, in someexamples, may be made of silicone, resin, glass, glass fiber, polyvinylchloride, polyethylene, polyamide, polyethylene, polypropylene,acrylonitrile butadiene, polycarbonate, polyetheretheketone (PEEK),polysulfone and polyurethane, co-polymers of these and other polymers.Exemplary materials include crystal styrene and cyclic olephinco-polymers. These structures or portions thereof can be created throughvarious techniques, e.g., injection molding, creation of structurallayers that are fused, etc. Polycarbonate and cyclic olephin polymersare preferred materials.

The electroporation chamber 516, in some embodiments, is generallycylindrical in shape. In other embodiments, the electroporation chamber516 may be rectangular, conical, or square.

The filter 506 can be fashioned, in some examples, from porous plastics,hydrophobic polyethylene, cotton, or glass fibers. Preferably, thefilter 506 is composed of a low-cost material such as porous plastics.The filter may range between 0.2-500 mm in width, 0.2-500 mm in height,and 1-3,000 μl in volume; between 0.3-250 mm in width, 20-200 mm inheight, and 50-2,500 μl in volume; or between 0.5-150 mm in width,0.2-80 mm in height, and 10-2,000 μl in volume.

The engagement member 504 is configured to have a dimension that iscompatible with the liquid handling device used in the electroporationinstrument.

The components of the electroporation devices may be manufacturedseparately and then assembled, or certain components of theelectroporation devices may be manufactured or molded as a singleentity, with other components added after molding. For example, thesipper, electroporation walls, and housing may be manufactured or moldedas a single entity, with the electrodes, filter, engagement member lateradded to the single entity to form the electroporation module.Similarly, the electroporation walls and housing may be manufactured asa single entity, with the sipper, electrodes, filter, engagement memberadded to the electroporation module after molding. Other combinations ofintegrated and non-integrated components are possible.

The electrodes 512 can be formed from a metal, such as copper, titanium,aluminum, brass, silver, rhodium, gold or platinum, or graphite, capableof withstanding application of an electric field. For example, anapplied electric field can destroy electrodes made from of metals likealuminum. If a multiple use electroporation device is desired, theelectrode plates can be coated with metals resistant to electrochemicalcorrosion. Conductive coatings like noble metals, e.g., gold, can beused to protect the electrode plates. In a particular example, theelectroporation cuvette may include a first metal electrode and a secondmetal electrode made from titanium covered with a layer of gold.Conversely, if the electroporation device 500 is designed for single use(e.g., disposable), less expensive metals such as aluminum may be used.

In one embodiment, the distance between the electrodes may be between0.3 mm and 10 mm. In another embodiment, the distance between theelectrodes may be between 1 mm and 20 mm, or 1 mm to 10 mm, or 2 mm to 5mm. The inner diameter of the electroporation chamber may be between 0.1mm and 10 mm. To avoid different field intensities between theelectrodes, the electrodes should by arranged in parallel with aconstant distance to each other over the whole surface of theelectrodes. Preferably, the first metal electrode and the second metalelectrode are separated by a distance of 2-4 mm in a parallelarrangement with variations in distance less than +/−20 μm. Furthermore,the surface of the electrodes should be as smooth as possible withoutpin holes or peaks. Electrodes having a roughness Rz of 1 to 10 μm arepreferred. In other embodiments, the electroporation device includes atleast one additional electrode which applies a ground potential to,e.g., the sipper portion of the electroporation device.

Although illustrated as a single unit device 500, in other embodiments,the electroporation module includes multiple electroporation units. Eachelectroporation unit may be configured to electroporate cell samplevolumes of between 1 μl to 20 ml. For example, differing volumecapacities of electroporation units may be available in a multi-unitelectroporation device.

In a multi-unit electroporation module, in some embodiments, theelectrodes are independent, standalone elements. In other embodiments, amulti-unit electroporation device may include electrodes arranged suchthat electroporation cuvettes in adjacent electroporation units shareelectrodes. Such multi-unit electroporation devices may include, e.g., 2or more electroporation units, 4 or more electroporation units, 8 ormore electroporation units, 16 or more electroporation units, 32 or moreelectroporation units, 48 or more electroporation units, 64 or moreelectroporation units, or even 96 or more electroporation unitspreferably in an automated device. Where multiple parallel devices areemployed, typically like volumes are used in each unit.

Although example dimensions are provided, the dimensions, of course,will vary depending on the volume of the cell sample and thecontainer(s) that are used to contain the cells and/or material to beelectroporated.

In preferred embodiments, the transformation module includes at leastone flow-through electroporation device having a housing with anelectroporation chamber, a first electrode and a second electrodeconfigured to engage with an electric pulse generator. In someimplementations, the flow-through electroporation devices are configuredto mate with a replaceable cartridge such as the cartridges 104, 106 ofFIG. 1A (e.g., transformation module 110 c), by which electricalcontacts engage with the electrodes of the electroporation device. Incertain embodiments, the electroporation devices are autoclavable and/ordisposable, are packaged with reagents in the reagent cartridge, and/ormay be removable from the reagent cartridge. The electroporation devicemay be configured to electroporate cell sample volumes between 1 μl to 2ml, 10 μl to 1 ml, 25 μl to 750 μl, or 50 μl to 500 μl. The cells thatmay be electroporated with the disclosed electroporation devices includemammalian cells (including human cells), plant cells, yeasts, othereukaryotic cells, bacteria, archaea, and other cell types.

The reagent cartridges for use in the automated multi-module cellprocessing systems (e.g., cartridge 104 of FIG. 1A), in someembodiments, include one or more electroporation devices (e.g.,electroporation module 110 c of FIG. 1A), preferably flow-throughelectroporation devices. FIG. 5B is a bottom perspective view of a set530 of six co-joined flow-through electroporation devices (e.g., unitsor modules) 532 a-f that may be part of a reagent cartridge, and FIG. 5Cis a top perspective view of the same. The cartridge may include one tosix or more flow-through electroporation units 532 a-f arranged on asingle substrate 534. Each of the six flow-through electroporation units532 a-f have corresponding wells 536 a-f that define cell sample inletsand wells 538 a-f (see FIG. 5C) that define cell sample outlets.Additionally, as seen in FIG. 5B, each electroporation unit 532 a-fincludes a respective inlet 540 a-f, a respective outlet 542 a-f, arespective flow channel 544 a-f, and two electrodes 546 a-f on eitherside of a constriction in the respective flow channel 544 a-f of eachflow-through electroporation unit 532 a-f.

Once the six flow-through electroporation units 532 a-f are fabricated,in some embodiments, they can be separated from one another along thescore lines separating each unit from the adjacent unit (i.e., “snappedapart”) and used one at a time, or alternatively in other embodimentstwo or more flow-through electroporation units 532 a-f can be used inparallel, in which case those two or more units preferably remainconnected along the score lines.

Generally speaking, microfluidic electroporation—using cell suspensionvolumes of less than approximately 10 ml and as low as 1 μl—allows moreprecise control over a transfection or transformation process andpermits flexible integration with other cell processing tools comparedto bench-scale electroporation devices. Microfluidic electroporationthus provides unique advantages for, e.g., single cell transformation,processing and analysis; multi-unit electroporation deviceconfigurations; and integrated, automatic, multi-module cell processingand analysis.

The flow-through electroporation devices included in the reagentcartridges can achieve high efficiency cell electroporation with lowtoxicity. In specific embodiments of the flow-through electroporationdevices of the disclosure the toxicity level of the transformationresults in greater than 10% viable cells after electroporation,preferably greater than 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 70%, 75%, 80%, 85%, 90%, or even 95% viable cells followingtransformation, depending on the cell type and the nucleic acids beingintroduced into the cells.

After transformation, the cells are allowed to recover under conditionsthat promote the genome editing process that takes place as a result ofthe transformation and expression of the introduced nucleic acids in thecells.

Method for Automated Multi-Module Cell Processing

FIG. 9 is a flow chart of an example method 900 for using an automatedmulti-module cell processing system such as the systems illustrated inFIGS. 1A-1B and 12A-12B. The processing system of FIG. 13, for example,may direct the processing stage of the method 900. For example, asoftware script may identify settings for each processing stage andinstructions for movement of a robotic handling system to perform theactions of the method 900. In some embodiments, a software instructionscript may be identified by a cartridge supplied to the automatedmulti-module cell processing instrument. For example, the cartridge mayinclude machine-readable indicia, such as a bar code or QR code,including identification of a script stored in a memory of the automatedmulti-module cell processing instrument (e.g., such as memory 1302 ofFIG. 13). In another example, the cartridge may contain a downloadablescript embedded in machine-readable indicia such as a radio frequency(RF) tag. In other embodiments, the user may identify a script, forexample through downloading the script via a wired or wirelessconnection to the processing system of the automated multi-module cellprocessing instrument or through selecting a stored script through auser interface of the automated multi-module cell processing instrument.In a particular example, the automated multi-module cell processinginstrument may include a touch screen interface for submitting usersettings and activating cell processing.

In some implementations, the method 900 begins with transferring cellsto a growth module (902). The growth module, for example, may be thegrowth module 800 described in relation to FIGS. 8A through 8F. In aparticular example, the processing system 120 may direct the robotichandling system 108 to transfer cells 106 to the growth module 110 a, asdescribed in relation to FIGS. 12A and 12B. In another example, asdescribed in relation to FIG. 1A, the cells may be transferred from oneof the cartridges 104, 106 to the growth modules 110 a, 110 b by therobotic handling system 108. In some embodiments, the growth vial maycontain growth media and be supplied, e.g., as part of a kit. In otherembodiments, the growth vial may be filled with medium transferred,e.g., via the liquid handling device, from a reagent container.

In some embodiments, prior to transferring the cells (e.g., from thereagent cartridge 104 or from a vial added to the instrument),machine-readable indicia may be scanned upon the vial or other containersituated in a position designated for cells to confirm that the vial orcontainer is marked as containing cells. Further, the machine-readableindicia may indicate a type of cells provided to the instrument. Thetype of cells, in some embodiments, may cause the instrument to select aparticular processing script (e.g., series of instructions for therobotic handling system and settings and activation of the variousmodules).

In some implementations, the cells are grown in the growth module to adesired optical density (904). For example, the processing system 126 ofFIGS. 1A-1B or processing system 1220 of FIGS. 12A-B may manage atemperature setting of the growth module 110 a for incubating the cellsduring the growth cycle. The processing system 126, 1220 may furtherreceive sensor signals from the growth module 110 a, 110 b indicative ofoptical density and analyze the sensor signals to monitor growth of thecells. In some embodiments, a user may set growth parameters formanaging growth of the cells. For example, temperature, and the degreeof agitation of the cells. Further, in some embodiments, the user may beupdated regarding growth process. The updates, in some examples, mayinclude a message presented on a user interface of the automatedmulti-module cell processing system, a text message to a user's cellphone number, an email message to an email account, or a messagetransmitted to an app executing upon a portable electronic device (e.g.,cell phone, tablet, etc.). Responsive to the messages, in someembodiments, the user may modify parameters, such as temperature, toadjust cell growth. For example, the user may submit updated parametersthrough a user interface of the automated multi-module cell processingsystem or through a portable computing device application incommunication with the automated multi-module cell processing system,such as a user interface 1100 of FIG. 11.

Although described in relation to optical density, in otherimplementations, cell growth within the growth module may be monitoredusing a different measure of cell density and physiological state suchas, in some examples, pH, dissolved oxygen, released enzymes, acousticproperties, and electrical properties.

In some implementations, upon reaching the desired optical density(904), the cells are transferred from the growth module to a filtrationmodule or cell wash and concentration module (906). The robotic handlingsystem 108 of FIGS. 1A-1B or 1208 of FIGS. 12A-12B, for example, maytransfer the cells from the growth module 1210 a to the filtrationmodule 1210 b. The filtration module, for example, may be designed torender the cells electrocompetent. Further, the filtration module may beconfigured to reduce the volume of the cell sample to a volumeappropriate for electroporation. In another example, the filtrationmodule may be configured to remove unwanted components, such as salts,from the cell sample. In some embodiments, the robotic handling system108 transfers a washing solution to the filtration module 1210 b forwashing the cells.

In some implementations, the cells are rendered electrocompetent andeluted in the filtration module or cell wash and concentration module(908). The cells may be eluted using a wash solution. For example, thecells may be eluted using reagents from a reagent supply. The filtrationmodule or cell wash and concentration module, for example, may besimilar to the filtration module 700 illustrated in FIGS. 7A and 7B. Asdiscussed above, numerous different methods can be used to wash thecells, including density gradient purification, dialysis, ion exchangecolumns, filtration, centrifugation, dilution, and the use of beads forpurification. In some aspects, the cell wash and concentration moduleutilizes a centrifugation device. In other aspects, the filtrationmodule utilizes a filtration instrument. In yet other aspects, the beadsare coupled to moieties that bind to the cell surface. These moietiesinclude but are not limited to antibodies, lectins, wheat germagglutinin, mutated lysozymes, and ligands. In other aspects, the cellsare engineered to be magnetized, allowing magnets to pellet the cellsafter wash steps. Mechanism of cell magnetization can include but notlimited to ferritin protein expression.

In some embodiments, the wash solution is transferred to the filtrationmodule prior to eluting. The robotic handling system 108 of FIGS.12A-12B, for example, may transfer the wash solution from a vial orcontainer situated in a position designated for wash solution. Prior totransferring the wash solution, machine-readable indicia may be scannedupon the vial or other container or reservoir situated in the positiondesignated for the wash solution to confirm the contents of the vial,container, or reservoir. Further, the machine-readable indicia mayindicate a type of wash solution provided to the instrument. The type ofwash solution, in some embodiments, may cause the system to select aparticular processing script (e.g., settings and activation of thefiltration module appropriate for the particular wash solution).

In other embodiments, the cells are eluted in a cell wash module of awash cartridge. For example, the eluted cells may be collected in anempty vessel of the wash cartridge 106 illustrated in FIG. 1A, and therobotic handling system 108 may transfer media from the reagentcartridge 104 (or a reagent well of the wash cartridge 10 b) to theeluted cells.

Once the cells have been rendered electrocompetent and suspended in anappropriate volume such as 50 μL to 10 mL, or 100 μL to 80 mL, or 150 μLto 8 mL, or 250 μL to 7 mL, or 500 μL to 6 mL, or 750 μL to 5 mL fortransformation by the filtration module (906), in some implementations,the cells are transferred to a transformation module (918). The robotichandling system 108 of FIGS. 1A-1B, for example, may transfer the cellsfrom the filtration module to the transformation module 110 c. Thefiltration module may be physically coupled to the transformationmodule, or these modules may be separate. In an embodiment such as theinstrument 100 of FIG. 1A having cartridge-based supplies, the cells maybe eluted to a reservoir within a cartridge, such as the reagentcartridge 104, prior to transferring to the transformation module.

In some implementations, nucleic acids are prepared outside of theautomated multi-module cell processing instrument. For example, anassembled vector or other nucleic acid assembly may be included as areagent by a user prior to running the transformation process and otherprocesses in the method 900.

However, in other implementations, nucleic acids are prepared by theautomated multi-module cell processing instrument. A portion of thefollowing steps 910 through 916, in some embodiments, are performed inparallel with a portion of steps 902 through 908. At least a portion ofthe following steps, in some embodiments, are performed before and/orafter steps 902 through 908.

In some implementations nucleic acids such as an editing oligonucleotideand a vector back bone, as well as, in some examples, enzymes and otherreaction components are transferred to a nucleic acid assembly module(910). The nucleic acid assembly module may be configured to perform oneor more of a wide variety of different nucleic acid assembly techniquesin an automated fashion. Nucleic acid assembly techniques that can beperformed in the nucleic acid assembly module may include, but are notlimited to, those assembly methods that use restriction endonucleases,including PCR, BioBrick assembly, Type IIS cloning (e.g., GoldenGateassembly), and Ligase Cycling Reaction. In other examples, the nucleicacid assembly module may perform an assembly technique based on overlapsbetween adjacent parts of the nucleic acids, such as Gibson Assembly®,CPEC, SLIC, Ligase Cycling etc. Additional example assembly methods thatmay be performed by the nucleic acid assembly module include gap repairin yeast, gateway cloning and topoisomerase-mediated cloning. Thenucleic acid assembly module, for example, may be the nucleic acidassembly module 400 described in relation to FIG. 4. In a particularexample, the processing system 120 may direct the robotic handlingsystem 1208 to transfer nucleic acids 1206 to the nucleic acid assemblymodule 1210 e, as described in relation to FIG. 12B. In another example,as described in relation to FIG. 1A, the nucleic acids may betransferred from one of the cartridges 104, 106 to a nucleic acidassembly module by the robotic handling system 108.

In some embodiments—prior to transferring each of the nucleic acidsamples, the enzymes, and other reaction components—machine-readableindicia may be scanned upon the vials or other containers situated inpositions designated for these materials to confirm that the vials orcontainers are marked as containing the anticipated material. Further,the machine-readable indicia may indicate a type of one or more of thenucleic acid samples, the enzymes, and other reaction componentsprovided to the instrument. The type(s) of materials, in someembodiments, may cause the instrument to select a particular processingscript (e.g., series of instructions for the robotic handling system toidentify further materials and/or settings and activation of the nucleicacid assembly module).

In some embodiments, the nucleic acid assembly module is temperaturecontrolled depending upon the type of nucleic acid assembly used. Forexample, when PCR is utilized in the nucleic acid assembly module, themodule can have a thermocycling capability allowing the temperatures tocycle between denaturation, annealing and extension. When singletemperature assembly methods are utilized in the nucleic acid assemblymodule, the module can have the ability to reach and hold at thetemperature that optimizes the specific assembly process beingperformed.

Temperature control, in some embodiments, is managed by a processingsystem of the automated multi-module cell processing instrument, such asthe processing system 1310 of FIG. 13. These temperatures and theduration of maintaining the temperatures can be determined by apreprogrammed set of parameters (e.g., identified within the processingscript or in another memory space accessible by the processing system),or manually controlled by the user through interfacing with theprocessing system.

Once sufficient time has elapsed for the assembly reaction to takeplace, in some implementations, the nucleic acid assembly is transferredto a purification module (914). The processing system, for example, maymonitor timing of the assembly reaction based upon one or more of thetype of reaction, the type of materials, and user settings provided tothe automated multi-module cell processing instrument. The robotichandling system 108 of FIGS. 1A-1B or 12A-12B, for example, may transferthe nucleic acid assembly to the purification module through a sipper orpipettor interface. In another example, the robotic handling system 108of FIGS. 1A-1B or 12A-12B may transfer a vial containing the nucleicacid assembly from a chamber of the nucleic acid assembly module to achamber of the de-salt/purification module.

In some implementations, the nucleic acid assembly is de-salted andeluted at the purification module (916). The purification module, forexample, may remove unwanted components of the nucleic acid assemblymixture (e.g., salts, minerals, etc.). In some embodiments, thepurification module concentrates the assembled nucleic acids into asmaller volume that the nucleic acid assembly volume. Examples ofmethods for exchanging liquid following nucleic acid assembly includemagnetic beads (e.g., SPRI or Dynal (Dynabeads) by Invitrogen Corp. ofCarlsbad, Calif.), silica beads, silica spin columns, glass beads,precipitation (e.g., using ethanol or isopropanol), alkaline lysis,osmotic purification, extraction with butanol, membrane-based separationtechniques, filtration etc. For example, one or more micro-concentratorsfitted with anisotropic, hydrophilic-generated cellulose membranes ofvarying porosities may be used. In another example, thede-salt/purification module may process a liquid sample including anucleic acid and an ionic salt by contacting the mixture with an ionexchanger including an insoluble phosphate salt, removing the liquid,and eluting nucleic acid from the ion exchanger.

In an illustrative embodiment, the nucleic acid assembly may be combinedwith magnetic beads, such as SPRI beads, in a chamber of a purificationmodule. The nucleic acid assembly may be incubated at a set temperaturefor sufficient time for the nucleic acid assembly to bind to themagnetic beads. After incubation, a magnet may be engaged proximate tothe chamber so that the nucleic acid assembly can be washed and eluted.An illustrative example of this process is discussed in relation to thecombination isothermal nucleic acid assembly and purification module ofFIG. 4.

Once the nucleic acid assembly has been eluted, the nucleic acidassembly, in some implementations, is transferred to the transformationmodule (918). The robotic handling system 108 of FIGS. 1A-1B or 12A-12B,for example, may transfer the nucleic acid assembly to thetransformation module through a sipper or pipettor interface to, e.g., acuvette-based electroporator module or a flow-through electroporatormodule, as described above. For example, the de-salted assembled nucleicacids, during the transfer, may be combined with the electrocompetentcells from step 908. In other embodiments, the transformation module mayaccept each of the electrocompetent cells and the nucleic acid assemblyseparately and enable the mixing (e.g., open one or more channels tocombine the materials in a shared chamber).

The cells may be transformed in the transformation module (920).Transformation may involve any art-recognized technique for introducingan exogenous nucleic acid sequence (e.g., DNA) into a target cell(either transformation or transfection), including, in some examples,electroporation, lipofection, optoporation, injection,microprecipitation, microinjection, liposomes, particle bombardment,sonoporation, laser-induced poration, bead transfection, calciumphosphate or calcium chloride co-precipitation, or DEAE-dextran-mediatedtransfection. In some embodiments, hybrid techniques that exploit thecapabilities of mechanical and chemical transfection methods can beused, such as magnetofection, a transfection methodology that combineschemical transfection with mechanical methods. In another example,cationic lipids may be deployed in combination with gene guns orelectroporators.

In some implementations, the transformation module uses electroporationto trigger uptake of the DNA material. A buffer or medium may betransferred to the transformation module and added to the cells so thatthe cells may be suspended in a buffer or medium that is favorable forcell survival during electroporation. Prior to transferring the bufferor medium, machine-readable indicia may be scanned upon the vial orother container or reservoir situated in the position designated for thebuffer or medium to confirm the contents of the vial, container, orreservoir. Further, the machine-readable indicia may indicate a type ofbuffer or medium provided to the instrument. The type of buffer ormedium, in some embodiments, may cause the instrument to select aparticular processing script (e.g., settings and activation of thetransformation module appropriate for the particular buffer or medium).For bacterial cell electroporation, low conductance mediums, such aswater or glycerol solutions, may be used to reduce the heat productionby transient high current. For yeast cells a sorbitol solution may beused. For mammalian cell electroporation, cells may be suspended in ahighly conductive medium or buffer, such as MEM, DMEM, IMDM, RPMI,Hanks', PBS, HBSS, HeBS and Ringer's solution. In a particular example,the robotic handling system 108 may transfer a buffer solution to thetransformation module 110 c from one of the cartridges 104, 106. Thetransformation module, for example, may be a flow-throughelectroporation module such as the electroporation modules described inrelation to FIGS. 5A and 5B. As described in relation to FIG. 1A andFIG. 5B, the transformation module may be a disposable flow-throughelectroporation module 110 c provided with the cartridge 104 of FIG. 1A.

In some implementations, the transformation module further prepares thecells for nucleic acid uptake. For example, bacterial cells may betreated with a sucrose or glycerol wash prior to addition of nucleicacids, and yeast cells may be treated with a solution of lithiumacetate, dithiotheitol (DTT) and TE buffer. In other implementationsinvolving preparation of cells for nucleic acid uptake, the filtrationmodule or another separate module (e.g., a cell wash module) may preparethe cells for nucleic acid update.

Once transformed, the cells are transferred to a secondgrowth/recovery/editing module (922). The robotic handling system 108 ofFIGS. 1A-1B or 12A-12B, for example, may transfer the transformed cellsto the second growth module through a sipper or pipettor interface. Inanother example, the robotic handling system 108 of 1A-1B or 12A-12B maytransfer a vial containing the transformed cells from a chamber of thetransformation module to a chamber of the second growth module.

The second growth module, in some embodiments, acts as a recoverymodule, allowing the cells to recover from the transformation process.In other embodiments, the cells may be provided to a separate recoverymodule prior to being transported to the second growth module. Duringrecovery, the second growth module allows the transformed cells touptake and, in certain aspects integrate the introduced nucleic acidsinto the genome of the cell. The second growth module may be configuredto incubate the cells at any user-defined temperature optimal for cellgrowth, preferably 25°, 30°, or 37° C.

In some embodiments, the second growth module behaves as a selectionmodule, selecting the transformed cells based on an antibiotic or otherreagent. In one example, the RNA-guided nuclease (RGN) protein system isused for selection to cleave the genomes of cells that have not receivedthe desired edit. The RGN protein system used for selection can eitherbe the same or different as the RGN used for editing. In the example ofan antibiotic selection agent, the antibiotic may be added to the secondgrowth module to enact selection. Suitable antibiotic resistance genesinclude, but are not limited to, genes such as ampicillin-resistancegene, tetracycline-resistance gene, kanamycin-resistance gene,neomycin-resistance gene, canavanine-resistance gene,blasticidin-resistance gene, hygromycin-resistance gene,puromycin-resistance gene, or chloramphenicol-resistance gene. Therobotic handling system 108 of FIGS. 1A-1B or 12A-12B, for example, maytransfer the antibiotic to the second growth module through a sipper orpipettor interface. In some embodiments, removing dead cell backgroundis aided using lytic enhancers such as detergents, osmotic stress byhypnotic wash, temperature, enzymes, proteases, bacteriophage, reducingagents, or chaotropes. The processing system 1310 of FIG. 13, forexample, may alter environmental variables, such as temperature, toinduce selection, while the robotic handling system 108 of FIGS. 1A-1Bor 12A-12B may deliver additional materials (e.g., detergents, enzymes,reducing agents, etc.) to aid in selection. In other embodiments, cellremoval and/or media exchange by filtration is used to reduce dead cellbackground.

In further embodiments, in addition to or as an alternative to applyingselection, the second growth module serves as an editing module,allowing for genome editing in the transformed cells. Alternatively, inother embodiments the cells post-recovery and selection (if performed)are transferred to a separate editing module. As an editing module, thesecond growth module induces editing of the cells' genomes, e.g.,through expression of the introduced nucleic acids. Expression of thenuclease may involve one or more of chemical, light, viral, ortemperature induction. The second growth module, for example, may beconfigured to heat or cool the cells during a temperature inductionprocess. In a particular illustration, the cells may be induced byheating at 42° C.-50° C. Further to the illustration, the cells may thenbe are cooled to 0-10° C. after induction. In the example of chemical orviral induction, an inducing agent may be transferred to the secondgrowth module to induce editing. If an inducible nuclease was introducedto the cells, during editing, the inducible nuclease is induced throughintroduction of an inducer molecule, such as the inducer molecule 1224described in relation to FIG. 12A. The inducing agent or inducermolecule, in some implementations, is transferred to the second growthmodule by the robotic handling system 108 of FIGS. 1A-1B or 12A-12B(e.g., through a pipettor or sipper interface).

In some implementations, if no additional cell editing is desired (924),the cells may be transferred from the cell growth module to a storageunit for later removal from the automated multi-module cell processingsystem (926). The storage unit, for example, may include the storageunit 114 of FIGS. 12A-12B. The robotic handling system 108 of FIGS.1A-1B or 12A-12B, for example, may transfer the cells to the storageunit 114 through a sipper or pipettor interface. In another example, therobotic handling system 108 of FIGS. 1A-1B or 12A-12B may transfer avial containing the cells from a chamber of the second growth module toa vial or tube within the storage unit.

In some implementations, if additional cell editing is desired (924),the cells may be transferred to the same or a different filtrationmodule and rendered electrocompetent (908). Further, in someembodiments, a new assembled nucleic acid sample may be prepared by thenucleic acid assembly module at this time. Prior to recursive editing,in some embodiments, the automated multi-module cell processinginstrument may require additional materials (e.g., replacementcartridges) be supplied by the user.

The steps may be the same or different during the second round ofediting. For example, in some embodiments, upon a subsequent executionof step 904, a selective growth medium is transferred to the growthmodule to enable selection of edited cells from the first round ofediting. The robotic handling system 108 of FIGS. 1A-B or 12A-B, forexample, may transfer the selective growth medium from a vial orcontainer in a reagent cartridge situated in a position designated forselective growth medium. Prior to transferring the selective growthmedium, machine-readable indicia may be scanned upon the vial or othercontainer or reservoir situated in the position designated for theselective growth medium to confirm the contents of the vial, container,or reservoir. Further, the machine-readable indicia may indicate a typeof selective growth medium provided to the instrument. The type ofselective growth medium, in some embodiments, may cause the instrumentto select a particular processing script (e.g., settings and activationof the growth module appropriate for the particular selective growthmedium). Particular examples of recursive editing workflows aredescribed in relation to FIGS. 10A through 10C.

In some implementations, the method 900 can be timed to requestmaterials and/or complete the editing cycle in coordination with auser's schedule. For example, the automated multi-module cell processinginstrument may provide the user the ability to schedule completion ofone or more cell processing cycles (e.g., one or more recursive edits)such that the method 900 is enacted with a goal of completion at theuser's preferred time. The time scheduling, for example, may be setthrough a user interface, such as the user interface 1316 of FIG. 13. Ina particular illustration, a user may set completion of a first cycle to4:00 PM so that the user can supply additional cartridges of materialsto the automated multi-module cell processing instrument to enableovernight processing of another round of cell editing.

In some implementations, throughout the method 900, the automatedmulti-module cell processing instrument may alert the user to itscurrent status. For example, the user interface 1316 of FIG. 13 maypresent a graphical indication of the present stage of processing. In aparticular example, a front face of the automated multi-module callprocessing instrument may be overlaid with a user interface (e.g., touchscreen) that presents an animated graphic depicting present status ofthe cell processing. The user interface may further present any userand/or default settings associated with the current processing stage(e.g., temperature setting, time setting, etc.).

Although illustrated as a particular series of operations, in otherembodiments, more or fewer steps may be included in the method 900. Forexample, in some embodiments, prior to engaging in each round ofediting, the contents of reservoirs, cartridges, and/or vials may bescreened to confirm appropriate materials are available to proceed withprocessing. For example, in some embodiments, one or more imagingsensors (e.g., barcode scanners, cameras, etc.) may confirm contents atvarious locations within the housing of the automated multi-module cellprocessing instrument. In one example, multiple imaging sensors may bedisposed within the housing of the automated multi-module cellprocessing instrument, each imaging sensor configured to detect one ormore materials (e.g., machine-readable indicia such as barcodes or QRcodes, shapes/sizes of materials, etc.). In another example, at leastone imaging sensor may be moved by the robotic handling system tomultiple locations to detect one or more materials. In furtherembodiments, one or more weight sensors may detect presence or absenceof disposable or replaceable materials. In an illustrative example, thetransfer tip supply holder 116 may include a weight sensor to detectwhether or not tips have been loaded into the region. In anotherillustrative example, an optical sensor may detect that a level ofliquid waste has reached a threshold level, requiring disposal prior tocontinuation of cell processing. Requests for additional materials,removal of waste supplies, or other user interventions (e.g., manualcleaning of one or more elements, etc.), in some implementations, arepresented on a graphical user interface of the automated multi-modulecell processing instrument. The automated multi-module cell processinginstrument, in some implementations, contacts the user with requests fornew materials or other manual interventions, for example through asoftware app, email, or text message.

Workflows for Cell Processing in an Automated Multi-Module CellProcessing Instrument

The automated multi-module cell processing instrument is designed toperform a variety of cell processing workflows using the same modules.For example, source materials, in individual containers or in cartridgeform, may differ and the corresponding instructions (e.g., softwarescript) may be selected accordingly, using the same basicinstrumentation and robotic handling system; that is, the multi-modulecell processing system can be configured to perform a number ofdifferent workflows for processing cell samples and different types ofcell samples. In embodiments, a same workflow may be performediteratively to recursively edit a cell sample. In other embodiments, acell sample is recursively edited, but the workflow may change fromiteration to iteration.

FIGS. 10A through 10C illustrate example workflows that may be performedusing an automated multi-module cell processing instrument including twocell growth modules 1002, 1008, two filtration modules 1004 and 1010,and a flow-through electroporation module 1006. Although described asseparate growth modules 1002, 1008 and filtration modules 1004, 1010,each may instead be designed as a dual module. For example, a dualgrowth module, including growth modules 1002 and 1008, may include dualrotating growth vials sharing some circuitry, controls, and a powersource and disposed in a same housing. Similarly, a dual filtrationmodule may include filtration modules 1004 and 1010, including twoseparate filters and liquid supply tubes but sharing circuitry,controls, a power source, and a housing. The modules 1002, 1004, 1006,1008, and 1010, for example, may be part of the instrument 100 describedin relation to FIGS. 1A and 1B.

Turning to FIG. 10A, a flow diagram illustrates a first bacteria genomeediting workflow 1000 involving two stages of processing havingidentical processing steps, resulting in two edits to a cell stock 1012.Each stage may operate based upon a different cartridge of sourcematerials. For example, a first cartridge may include a first oligolibrary 1014 a and a first sgRNA backbone 1016 a. A second cartridge,introduced into the automated multi-module cell processing instrumentbetween processing stages or prior to processing but in a differentposition than the first cartridge, may include a second oligo library1014 b and a second sgRNA backbone 1016 b. Each cartridge may beconsidered as a “library cartridge” for building a library of editedcells. The cell stock 1012, in some embodiments, is included in thefirst library cartridge. The cell stock 1012 may be supplied within akit including the two cartridges. Alternatively, a user may add acontainer (e.g., vial or tube) of the cell stock 1012 to a purchasedcartridge.

The workflow 1000, in some embodiments, is performed based upon a scriptexecuted by a processing system of the automated multi-module cellprocessing instrument, such as the processing system 1310 of FIG. 13.The script, in a first example, may be accessed via a machine-readablemarker or tag added to the first cartridge. In some embodiments, eachprocessing stage is performed using a separate script. For example, eachcartridge may include an indication of a script or a script itself forprocessing the contents of the cartridge.

In some implementations, the first stage begins with introducing thecell stock 1012 into the first growth module 1002 for inoculation,growth, and monitoring (1018 a). In one example, a robotic handlingsystem adds a vial of the cell stock 1012 to medium contained in therotating growth vial of the first growth module 1002. In anotherexample, the robotic handling system pipettes cell stock 1012 from thefirst cartridge and adds the cell stock 1012 to the medium contained inthe rotating growth vial. The cells may have been maintained at atemperature of 4° C. at this point. In a particular example, 20 ml ofcell stock may be grown within a rotating growth vial of the firstgrowth module 1002 at a temperature of 30° C. to an OD of 0.50. The cellstock 1012 added to the first growth module 1002 may be monitored overtime until 0.50 OD is sensed via automated monitoring of the growthvial. Monitoring may be periodic or continuous. This may take, forexample, around 900 minutes (estimated), although the exact time dependsupon detection of the desired OD.

In some implementations, after growing the cells to the desired OD, aninducer is added to the first growth module 1002 for inducing the cells.In a particular example, 100 μl of inducer may be added, and the growthmodule 1002 may bring the temperature of the mixture up to 42° C. andhold for 15 minutes.

The cell stock 1012, after growth and induction, is transferred to thefirst filtration module 1004, in some implementations, for rendering thecells electrocompetent (1020 a) and to reduce the volume of the cellsfor transformation. In one example, a robotic handling system moves thevial of the cell stock 1012 from the rotating growth vial of the firstgrowth module 1002 to a vial holder of the first filtration module 1004.In another example, the robotic handling system pipettes cell stock 1012from the rotating growth vial of the first growth module 1002 anddelivers it to the first filtration module 1004. For example, thedisposable pipetting tip used to transfer the cell stock 1012 to thefirst growth module 1002 may be used to transfer the cell stock 1012from the first growth module 1002 to the first filtration module 1004.In some embodiments, prior to transferring the cell stock 1012 from thefirst growth module 1002 to the first filtration module 1004, the firstgrowth module 1002 is cooled to 4° C. so that the cell stock 1012 issimilarly reduced to this temperature. In a particular example, thetemperature of the first growth module 1002 may be reduced to about 4°C. over the span of about 8 minutes, and the growth module 1002 may holdthe temperature at 4° C. for about 15 minutes to ensure reduction intemperature of the cell stock 1012.

Prior to transferring the cell stock, in some implementations, a filterof the first filtration module 1004 is pre-washed using a wash solution.The wash solution, for example, may be supplied in a wash cartridge,such as the cartridge 1006 described in relation to FIG. 1A. The firstfiltration module 1004, for example, may be fluidly connected to thewash solution of the wash cartridge, as described in relation to FIG.7A.

The first filtration module 1004, for example, may be part of a dualfiltration module such as the filtration module 750 described inrelation to FIGS. 7B and 7C. In a particular example, the firstfiltration module 1004 may be maintained at 4° C. during the washing andeluting process while transferring cell materials between an elutionvial and the first filtration module 1004.

In some implementations, upon rendering the cells electrocompetent atthe filtration module 1004, the cell stock 1012 is transferred to atransformation module 1006 (e.g., flow-through electroporation module)for transformation. In one example, a robotic handling system moves thevial of the cell stock 1012 from the vial holder of the first filtrationmodule 1004 to a reservoir of the flow-through electroporation module1006. In another example, the robotic handling system pipettes cellstock 1012 from the first filtration module 1002 or a temporaryreservoir and delivers it to the first filtration module 1004. In aparticular example, 400 μl of the concentrated cell stock 1012 from thefirst filtration module 1004 is transferred to a mixing reservoir priorto transfer to the transformation module 1006. For example, the cellstock 1012 may be transferred to a reservoir in a cartridge for mixingwith the assembled nucleic acids, then transferred by the robotichandling system using a pipette tip. In a particular example, thetransformation module is maintained at 4° C. The cell stock 1012 may betransformed, in an illustrative example, in about four minutes.

While the cells are growing and/or rendered electrocompetent, in someimplementations, a first oligo library 1014 a and the sgRNA backbone1016 a are assembled using an isothermal nucleic acid assembly processto create assembled nucleic acids in an isothermal nucleic acid assemblymaster mix (1022 a). The assembled nucleic acids may be created at somepoint during the first processing steps 1018 a, 1020 a of the firststage of the workflow 1000. Alternatively, assembled nucleic acids maybe created in advance of beginning the first processing step 1018.

In some embodiments, the nucleic acids are assembled using an isothermalnucleic acid assembly module of the automated multi-module cellprocessing instrument. For example, the robotic handling system may addthe first oligo library 1014 a and the sgRNA backbone 1016 a from alibrary vessel in the reagent cartridge in the automated multi-modulecell processing instrument to an isothermal nucleic acid assembly module(not illustrated), such as the nucleic acid assembly module 1210 gdescribed in relation to FIG. 12B. The nucleic acid assembly mix, forexample, may include in a particular example 50 μl Gibson Assembly®Master Mix, 25 μl vector backbone 1016 a, and 25 μl oligo 1014 a. Theisothermal nucleic acid assembly module may be held at room temperature.The assembly process may take about 30 minutes.

In other embodiments, the nucleic acids are assembled externally to themulti-module cell processing instrument and added as a source material.For example, a vial or tube of assembled nucleic acids may be added to areagent cartridge prior to activating the first step 1018 a of cellprocessing. In a particular example, 100 μl of assembled nucleic acidsare provided.

In some implementations, the assembled nucleic acids are purified (1024a). The assembled nucleic acids, for example, may be transferred by therobotic handling system from the isothermal nucleic acid assembly moduleto a purification module (not shown), such as the purification module1210 h of FIG. 12B. In other embodiments, the isothermal nucleic acidassembly module may include purification features (e.g., a combinationisothermal nucleic acid assembly and purification module). In furtherembodiments, the assembled nucleic acids are purified externally to themulti-module cell processing instrument and added as a source material.For example, a vial or tube of purified assembled nucleic acids may beadded to a reagent cartridge with the cell stock 1012 prior toactivating the first step 1018 a of cell processing.

In a particular example, 100 μl of assembled nucleic acids in isothermalnucleic acid assembly mix are purified. In some embodiments, magneticbeads are added to the isothermal nucleic acid assembly module, forexample 180 μl of magnetic beads in a liquid suspension may be added tothe isothermal nucleic acid assembly module by the robotic handlingsystem. A magnet functionally coupled to the isothermal nucleic acidassembly module may be activated and the sample washed in 200 μl ethanol(e.g., the robotic handling system may transfer ethanol to theisothermal nucleic acid assembly module). Liquid waste from thisoperation, in some embodiments, is transferred to a waste receptacle ofthe cartridge (e.g., by the robotic handling system using a same pipettetip as used in transferring the ethanol). At this point, the de-saltedassembled nucleic acids may be transferred to a holding container, suchas a reservoir of the cartridge. The desalted assembled nucleic acidsmay be held, for example at a temperature of 4° C. until cells are readyfor transformation. In a particular example, 100 μl of the assemblednucleic acids may be added to the 400 μl of the concentrated cell stock1012 in the mixing reservoir prior to transfer to the transformationmodule 1006. In some embodiments, the purification process may takeabout 16 minutes.

In some implementations, the assembled nucleic acids and cell stock 1012are added to the flow-through electroporation module 1006 and the cellstock 1012 is transformed (1026 a). The robotic handling system, forexample, may transfer the mixture of the cell stock 1012 and assemblednucleic acids to the flow-through electroporation module 1006 from amixing reservoir, e.g., using a pipette tip or through transferring avial or tube. In some embodiments, a built-in flow-throughelectroporation module such as the flow-through electroporation modules500 of FIG. 5A is used to transform the cell stock 1012. In otherembodiments, a cartridge-based electroporation module such as theflow-through electroporation module 530 of FIG. 5B is used to transformthe cell stock 1012. The electroporation module 1006, for example, maybe held at a temperature of 4° C. The electroporation process, in anillustrative example, may take about four minutes.

The transformed cell stock 1012, in some implementations, is transferredto the second growth module 1008 for recovery (1028 a). In a particularexample, transformed cells undergo a recovery process in the secondgrowth module 1008 at a temperature of 30° C. The transformed cells, forexample, may be maintained in the second growth module 1008 for about anhour for recovery.

In some implementations, a selective medium is transferred to the secondgrowth vial (not illustrated), and the cells are left to incubate for afurther period of time in a selection process. In an illustrativeexample, an antibiotic may be transferred to the second growth vial, andthe cells may incubate for an additional two hours at a temperature of30° C.

After recovery, the cells may be ready for either another round ofediting or for storage in a vessel, e.g., for further experimentsconducted outside of the automated cell processing environment.Alternatively, a portion of the cells may be transferred to a storageunit as cell library output, while another portion of the cells may beprepared for a second round of editing.

In some implementations, in preparation for a second round of editing,the transformed cells are transferred to the second filtration module1010 for media exchange and filtering (1030 a). Prior to transferringthe transformed cell stock, in some implementations, a filter of thesecond filtration module 1004 is pre-washed using a wash solution. Thewash solution, for example, may be supplied in a wash cartridge, such asthe cartridge 1006 described in relation to FIG. 1A. The secondfiltration module 1010, for example, may be fluidly connected to thewash solution of the wash cartridge, as described in relation to FIG.7A.

The second filtration module 1010, for example, may be part of a dualfiltration module such as the filtration module 750 described inrelation to FIGS. 7B and 7C. In a particular example, the secondfiltration module 1010 may be maintained at 4° C. during the washing andeluting process while transferring cell materials between an elutionvial and the second filtration module 1010. The output of thisfiltration process, in a particular example, is deposited in a vial ortube to await further processing, e.g., transfer to a transformationmodule. The vial or tube may be maintained in a storage unit at atemperature of 4° C.

The first stage of processing may take place during a single day. In anillustrative embodiment, the first stage of processing is estimated totake under 19 hours to complete (e.g., about 18.7 hours). At this pointin the workflow 1000, in some implementations, new materials aremanually added to the automated multi-module cell processing instrument.For example, a new reagent cartridge may be added. Further, a new washcartridge, replacement filters, and/or replacement pipette tips may beadded to the automated multi-module cell processing instrument at thispoint. Further, in some embodiments, the filter module may undergo acleaning process and/or the solid and liquid waste units may be emptiedin preparation for the next round of processing. In yet otherembodiments, the reagent cartridges may provide reagents for two or morecycles of editing.

In some implementations, the second round of editing involves the samemodules 1002, 104, 1006, 1008, and 1010, the same processing steps 1018,1020, 1022, 1024, 1026, 1028, and 1030, and the same temperature andtime ranges as the first processing stage described above. For example,the second oligo library 1014 b and the second sgRNA backbone 1016 b maybe used to edit the transformed cells in much the same manner asdescribed above. Although illustrated as a two-stage process, in otherembodiments, up to two, four, six, eight, or more recursions may beconducted to continue to edit the same cell stock 1012.

In other implementations, turning to FIG. 10B, a workflow 1040 involvesthe same modules 1002, 1004, 1006, 1008, and 1010 as well as the sameprocessing steps 1018, 1020, 1022, 1024, 1026, 1028, and 1030 for thefirst stage of process. However, unlike the workflow 1000 of FIG. 10A, asecond stage of the workflow 1040 of FIG. 10B involves a curing steps.“Curing” is a process in which a vector—for example the editing vectorused in the prior round of editing, the “engine” vector comprising theexpression sequence for the nuclease, or both—are eliminated from thetransformed cells. Curing can be accomplished by, e.g., cleaving theediting vector using a curing plasmid thereby rendering the editingand/or engine vector nonfunctional (exemplified in the workflow of FIG.10b ); diluting the vector in the cell population via cell growth (thatis, the more growth cycles the cells go through, the fewer daughtercells will retain the editing or engine vector(s)) (not shown), or by,e.g., utilizing a heat-sensitive origin of replication on the editing orengine vector (not shown). In one example, a “curing plasmid” may becontained within the reagent cartridge of the automated instrument, oradded manually to the instrument prior to the second stage ofprocessing. As with the workflow 1000, in some embodiments, the workflow1040 is performed based upon a script executed by a processing system ofthe automated multi-module cell processing instrument, such as theprocessing system 1310 of FIG. 13. The script, in a first example, maybe accessed via a machine-readable marker or tag added to the firstcartridge. In some embodiments, each processing stage is performed usinga separate script. For example, each cartridge may include an indicationof a script or a script itself for processing the contents of thecartridge. In this manner, for example, the second stage, involving thecuring cartridge, may be performed using a script designed for thesettings (e.g., temperatures, times, material quantities, etc.)appropriate for curing. The conditions for curing will depend on themechanism used for curing; that is, in this example, how the curingplasmid cleaves the editing and/or engine plasmid.

In some implementations, the second stage of the workflow 1040 begins byreceiving first-edited cells from the first stage of the workflow 1040at the first growth module 1002. For example, the first-edited cells mayhave been edited using a cell stock 1042, an oligo library 1044, and ansgRNA backbone 1046 through applying the steps 1018, 1020, 1022, 1024,1026, 1028, and 1030 as described in relation to the workflow 1000 ofFIG. 10A. The first-edited cell stock 1042, for example, may betransferred to the first growth module 1002 by a robotic handlingsystem. In one example, a robotic handling system adds a vial of thefirst-edited cell stock 1042 to a rotating growth vial of the firstgrowth module 1002. In another example, the robotic handling systempipettes first-edited cell stock 1042 from a receptacle of a storageunit and adds the cell stock 1042 to the rotating growth vial. The cellsmay have been maintained at a temperature of 4° C. at this point.

In some implementations, the first-edited cells are inoculated, grown,and monitored in the first growth module 1002 (1018 d). In a particularexample, an aliquot of the first-edited cell stock 1042 may betransferred to a rotating growth vial containing, e.g., 20 mL of growthmedium at a temperature of 30° C. to an OD of 0.50. The cell stock 1042added to the first growth module 1002 may be monitored over time until0.50 OD is sensed via the automated monitoring. Monitoring may beperiodic or continuous. This may take, for example, around 900 minutes(estimated), although the exact time depends upon detection of thedesired OD.

In some implementations, after growing to the desired OD, an inducer isadded to the first growth module 1002 for inducing the cells. In aparticular example, 100 μl of inducer may be added, and the growthmodule 1002 may bring the temperature of the mixture up to 42° C. andhold for 15 minutes.

The first-edited cell stock 1042, after growth and induction, istransferred to the first filtration module 1004, in someimplementations, for rendering the first-edited cells electrocompetent(1020 d). In one example, a robotic handling system moves the vial ofthe first-edited cell stock 1042 from the rotating growth vial of thefirst growth module 1002 to a vial holder of the first filtration module1004. In another example, the robotic handling system pipettesfirst-edited cell stock 1042 from the rotating growth vial of the firstgrowth module 1002 and delivers it to the first filtration module 1004.For example, the disposable pipetting tip used to transfer thefirst-edited cell stock 1042 to the first growth module 1002 may be usedto transfer the cell stock 1042 from the first growth module 1002 to thefirst filtration module 1004. In some embodiments, prior to transferringthe cell stock 1042 from the first growth module 1002 to the firstfiltration module 1004, the first growth module 1002 is cooled to 4° C.so that the cell stock 1042 is similarly reduced to this temperature. Ina particular example, the temperature of the first growth module 1002may be reduced to about 4° C. over the span of about 8 minutes, and thegrowth module 1002 may hold the temperature at 4° C. for about 15minutes to ensure reduction in temperature of the cell stock 1012.

Prior to transferring the first-edited cell stock 1042 to the filtrationmodule, in some implementations a filter of the first filtration module1004 is pre-washed using a wash solution. The wash solution, forexample, may be supplied in a wash cartridge, such as the cartridge 1006described in relation to FIG. 1A. The first filtration module 1004, forexample, may be fluidly connected to the wash solution of the washcartridge, as described in relation to FIG. 7A.

The first filtration module 1004, for example, may be part of a dualfiltration module such as the filtration module 750 described inrelation to FIGS. 7B and 7C. In a particular example, the firstfiltration module 1004 may be maintained at 4° C. during the washing andeluting process while transferring cell materials between an elutionvial and the first filtration module 1004.

In some implementations, upon rendering the first-edited cellselectrocompetent at the filtration module 1004 (1020 d), thefirst-edited cell stock 1042 is transferred to a transformation module1006 (e.g., flow-through electroporation module) for transformation. Inone example, a robotic handling system moves the vial of the cell stock1042 from the vial holder of the first filtration module 1004 to areservoir of the flow-through electroporation module 1006. In anotherexample, the robotic handling system pipettes cell stock 1042 from thefirst filtration module 1002 or a temporary reservoir and delivers it tothe first filtration module 1004. In a particular example, 400 μl of theconcentrated cell stock 1042 from the first filtration module 1004 istransferred to a mixing reservoir prior to transfer to thetransformation module 1006. For example, the cell stock 1042 may betransferred to a reservoir in a cartridge for mixing with a curingplasmid 1050, then mixed and transferred by the robotic handling systemusing a pipette tip. In a particular example, the transformation module1006 is maintained at 4° C. The cell stock 1042 may be transformed, inan illustrative example, in about four minutes.

The transformed cell stock 1042, in some implementations, is transferredto the second growth module 1008 for recovery/curing (1028 d). In aparticular example 20 ml of transformed cells undergo a recovery processin the second growth module 1008 at a temperature of 30° C. Thetransformed cells, for example, may be maintained in the second growthmodule 1008 for about an hour for recovery. If another round of editingis desired, the first editing plasmid or vector is cured. If anotherround of editing is not desired, the first editing plasmid and theengine plasmid may be cured.

After recovery and curing, the cells may be ready for either anotherround of editing or for storage to be used in further research outsidethe automated cell processing instrument. For example, a portion of thecells may be transferred to a storage unit as cell library output, whileanother portion of the cells may be prepared for a second round ofediting.

In some implementations, in preparation for a second round of editing,the transformed cells are transferred to the second filtration module1010 for media exchange and filtering (1030 d) containing glycerol forrendering the cells electrocompetent. Prior to transferring thetransformed cell stock, in some implementations, a filter of the secondfiltration module 1004 is pre-washed using a wash solution. The washsolution, for example, may be supplied in a wash cartridge, such as thecartridge 1006 described in relation to FIG. 1A. The second filtrationmodule 1010, for example, may be fluidly connected to the wash solutionof the wash cartridge, as described in relation to FIG. 7A.

The second filtration module 1010, for example, may be part of a dualfiltration module such as the filtration module 750 described inrelation to FIGS. 7B and 7C. In a particular example, the secondfiltration module 1010 may be maintained at 4° C. during the washing andeluting process while transferring cell materials between an elutionvial and the second filtration module 1010. The output of thisfiltration process, in a particular example, are electrocompetent cellsdeposited in a vial or tube to await further processing. The vial ortube may be maintained in a storage unit at a temperature of 4° C.

Turning to FIG. 10C, a flow diagram illustrates a yeast workflow 1060involving two stages of processing having identical processing steps,resulting in two edits to a cell stock 1062. Each stage may operatebased upon a different cartridge of source materials. For example, afirst cartridge may include a first oligo library 1070 a and a firstsgRNA back bone 1072 a. A second cartridge, introduced into theautomated multi-module cell processing instrument between processingstages or prior to processing but in a different position than the firstcartridge, may include a second oligo library 1070 b and a second sgRNAback bone 1072 b. Each cartridge may be considered as a “librarycartridge” for building a library of edited cells. Alternatively, a usermay add a container (e.g., vial or tube of the cell stock 1062 a to eachof the purchased cartridges included in a yeast cell kit.

The workflow 1060, in some embodiments, is performed based upon a scriptexecuted by a processing system of the automated multi-module cellprocessing system, such as the processing system 1310 of FIG. 13. Thescript, in a first example, may be accessed via a machine-readablemarker or tag added to the first cartridge. In some embodiments, eachprocessing stage is performed using a separate script. For example, eachcartridge may include an indication of a script or a script itself forprocessing the contents of the cartridge.

In some implementations, the first stage begins with introducing thecell stock 1062 into the first growth module 1002 for inoculation,growth, and monitoring (1018 e). In one example, a robotic handlingsystem adds a vial of the cell stock 1062 to a rotating growth vial ofthe first growth module 1002. In another example, the robotic handlingsystem pipettes cell stock 1062 from the first cartridge and adds thecell stock 1062 to the rotating growth vial. The cells may have beenmaintained at a temperature of 4° C. at this point. In a particularexample, 20 ml of cell stock may be grown within a rotating growth vialof the first growth module 1002 at a temperature of 30° C. to an OD of0.75. The cell stock 1012 added to the first growth module 1002 may beautomatically monitored over time within the growth module 1002 until0.75 OD is sensed via the automated monitoring. Monitoring may beperiodic or continuous.

In some implementations, an inducible expression system may be used.Thus, after growing to the desired OD, an inducer is added to the firstgrowth module 1002 for inducing the cells. The inducer could be a smallmolecule or a media exchange to a medium with a different sugar likegalactose.

The cell stock 1062, after growth and induction, is transferred to thefirst filtration module 1004, in some implementations, for exchangingmedia (1064 a). In one example, a robotic handling system moves the vialof the cell stock 1062 from the rotating growth vial of the first growthmodule 1002 to a vial holder of the first filtration module 1004. Inanother example, the robotic handling system pipettes cell stock 1062from the rotating growth vial of the first growth module 1002 anddelivers it to the first filtration module 1004. For example, thedisposable pipetting tip used to transfer the cell stock 1062 a to thefirst growth module 1002 may be used to transfer the cell stock 1062from the first growth module 1002 to the first filtration module 1004.In some embodiments, prior to transferring the cell stock 1062 from thefirst growth module 1002 to the first filtration module 1004, the firstgrowth module 1002 is cooled to 4° C. so that the cell stock 1062 issimilarly reduced to this temperature. In a particular example, thetemperature of the first growth module 1002 may be reduced to about 4°C. over the span of about 8 minutes, and the growth module 1002 may holdthe temperature at 4° C. for about 15 minutes to ensure reduction intemperature of the cell stock 1062. During media exchange, in anillustrative example, 0.4 ml of 1M sorbitol may be added to the cellstock 1062.

Prior to transferring the cell stock 1062, in some implementations, afilter of the first filtration module 1004 is pre-washed using a washsolution. The wash solution, for example, may be supplied in a washcartridge, such as the cartridge 1006 described in relation to FIG. 1A.The first filtration module 1004, for example, may be fluidly connectedto the wash solution of the wash cartridge, as described in relation toFIG. 7A.

The first filtration module 1004, for example, may be part of a dualfiltration module such as the filtration module 750 described inrelation to FIGS. 7B and 7C. In a particular example, the firstfiltration module 1004 may be maintained at 4° C. during the washing andeluting process while transferring cell materials between an elutionvial and the first filtration module 1004.

After the media exchange operation, in some implementations, the cellstock 1062 is transferred back to the first growth module 1002 forconditioning (1066 a). In one example, a robotic handling system movesthe vial of the cell stock 1062 from the first filtration module 1004 tothe first growth module 1002. In another example, the robotic handlingsystem pipettes cell stock 1062 from the first filtration module 1004and delivers it to the rotating growth vial of the first growth module1002. During conditioning, for example, 5 ml DTT/LIAc and 80 mM ofSorbitol may be added to the cell stock 1062. For example, the robotichandling system may transfer the DTT/LIAc and Sorbitol, individually orconcurrently, to the first growth module 1002. The cell stock 1062 maybe mixed with the DTT/LIAc and Sorbitol, for example, via the rotationof the rotating growth vial of the first growth module 1002. Duringconditioning, the cell stock 1062 may be maintained at a temperature of4° C.

In some implementations, after conditioning, the cell stock 1062 istransferred to the first filtration module 1004 for washing andpreparing the cells (1068). For example, the cells may be renderedelectrocompetent at this step. In one example, a robotic handling systemmoves the vial of the cell stock 1062 from the rotating growth vial ofthe first growth module 1002 to a vial holder of the first filtrationmodule 1004. In another example, the robotic handling system pipettescell stock 1062 from the rotating growth vial of the first growth module1002 and delivers it to the first filtration module 1004.

Prior to transferring the cell stock, in some implementations, a filterof the first filtration module 1004 is pre-washed using a wash solution.The wash solution, for example, may be supplied in a wash cartridge,such as the cartridge 1006 described in relation to FIG. 1A. The firstfiltration module 1004, for example, may be fluidly connected to thewash solution of the wash cartridge, as described in relation to FIG.7A. In other embodiments, the same filter is used for renderingelectrocompetent as the filter used for media exchange at step 1064 a.In some embodiments, 1M sorbitol is used to render the yeast cellselectrocompetent.

In some implementations, upon rendering electrocompetent at thefiltration module 1004, the cell stock 1062 is transferred to atransformation module 1006 (e.g., flow-through electroporation module)for transformation. In one example, a robotic handling system moves thevial of the cell stock 1062 from the vial holder of the first filtrationmodule 1004 to a reservoir of the flow-through electroporation module1006. In another example, the robotic handling system pipettes cellstock 1062 from the filtration module 1004 or a temporary reservoir anddelivers it to the first filtration module 1004. In a particularexample, 400 μl of the concentrated cell stock 1062 from the firstfiltration module 1004 is transferred to a mixing reservoir prior totransfer to the transformation module 1006. For example, the cell stock1062 may be transferred to a reservoir in a cartridge for mixing withthe nucleic acid components (backbone and editing oligonucleotide), thenmixed and transferred by the robotic handling system using a pipettetip. Because the backbone (vector) and editing oligonucleotide areassembled in the cells (in vivo), a nucleic acid assembly module is nota necessary component for yeast editing. In a particular example, thetransformation module is maintained at 4° C.

In some implementations, the nucleic acids to be assembled and the cellstock 1062 is added to the flow-through electroporation module 1006 andthe cell stock 1062 is transformed (1026 e). The robotic handlingsystem, for example, may transfer the mixture of the cell stock 1062 eand nucleic acid assembly to the flow-through electroporation module1006 from a mixing reservoir, e.g., using a pipette tip or throughtransferring a vial or tube. In some embodiments, a built-inflow-through electroporation module such as the flow-throughelectroporation modules 500 of FIG. 5A is used to transform the cellstock 1062 e. In other embodiments, a cartridge-based electroporationmodule such as the flow-through electroporation module 530 of FIG. 5B isused to transform the cell stock 1062 e. The electroporation module1006, for example, may be held at a temperature of 4° C.

The transformed cell stock 1062 e, in some implementations, istransferred to the second growth module 1008 for recovery (1028 a). In aparticular example, 20 ml of transformed cells undergo a recoveryprocess in the second growth module 1008.

In some implementations, a selective medium, e.g. an auxotrophic growthmedium or a medium containing a drug, is transferred to the secondgrowth vial (not illustrated), and the cells are left to incubate for afurther period of time in a selection process. In an illustrativeexample, an antibiotic may be transferred to the second growth vial, andthe cells may incubate for an additional two hours at a temperature of30° C.

After recovery, the cells may be ready for either another round ofediting or for storage in a cell library. For example, a portion of thecells may be transferred to a storage unit as cell library output (1076a), while another portion of the cells may be prepared for a secondround of editing (1078 a). The cells may be stored, for example, at atemperature of 4° C.

In some implementations, in preparation for a second round of editing,the transformed cells are transferred to the second filtration module1010 for media exchange (1078 a). Prior to transferring the transformedcell stock 1062 a, in some implementations, a filter of the secondfiltration module 1004 is pre-washed using a wash solution. The washsolution, for example, may be supplied in a wash cartridge, such as thecartridge 1006 described in relation to FIG. 1A. The second filtrationmodule 1010, for example, may be fluidly connected to the wash solutionof the wash cartridge, as described in relation to FIG. 7A.

The second filtration module 1010, for example, may be part of a dualfiltration module such as the filtration module 750 described inrelation to FIGS. 7B and 7C. In a particular example, the secondfiltration module 1010 may be maintained at 4° C. during the washing andeluting process while transferring cell materials between an elutionvial and the second filtration module 1010.

In some implementations during the filtration process, an enzymaticpreparation is added to lyse the cell walls of the cell stock 1062 a.For example, a yeast lytic enzyme such as Zylomase® may be added to lysethe cell walls. The yeast lytic enzyme, in a particular example, may beincubated in the cell stock 1026 a for between 5-60 minutes at atemperature of 30° C. The output of this filtration process, in aparticular example, is deposited in a vial or tube to await furtherprocessing. The vial or tube may be maintained in a storage unit at atemperature of 4° C.

The first stage of processing may take place during a single day. Atthis point of the workflow 1060, in some implementations, new materialsare manually added to the automated multi-module cell processinginstrument. For example, new cell stock 1062 b and a new reagentcartridge may be added. Further, a new wash cartridge, replacementfilters, and/or replacement pipette tips may be added to the automatedmulti-module cell processing system at this point. Further, in someembodiments, the filter module may undergo a cleaning process and/or thesolid and liquid waste units may be emptied in preparation for the nextround of processing.

In some implementations, the second round of editing involves the samemodules 1002, 104, 1006, 1008, and 1010, the same processing steps 1018,1064, 1066, 1026, 1028, and 1076 and/or 1078, and the same conditions(e.g., temperatures, time ranges, etc.) as the first processing stagedescribed above. For example, the second oligo library 1070 b and thesecond sgRNA backbone 1072 b may be used to edit a combination of thetransformed cells in much the same manner as described above. Althoughillustrated as a two-stage process, in other embodiments, up to two,three, four, six, eight, or more recursions may be conducted to continueto edit the cell stock 1062.

Example I: Fully-Automated Singleplex RGN-Directed Editing Run

Singleplex automated genomic editing using MAD7 nuclease wassuccessfully performed with an automated multi-module instrument of thedisclosure. See U.S. Pat. No. 9,982,279.

An ampR plasmid backbone and a lacZ_F172* editing cassette wereassembled via Gibson Assembly® into an “editing vector” in an isothermalnucleic acid assembly module included in the automated instrument.lacZ_F172 functionally knocks out the lacZ gene. “lacZ_F172*” indicatesthat the edit happens at the 172nd residue in the lacZ amino acidsequence. Following assembly, the product was de-salted in theisothermal nucleic acid assembly module using AMPure beads, washed with80% ethanol, and eluted in buffer. The assembled editing vector andrecombineering-ready, electrocompetent E. Coli cells were transferredinto a transformation module for electroporation. The transformationmodule comprised an ADP-EPC cuvette. See, e.g., U.S. Pat No. 62/551,069.The cells and nucleic acids were combined and allowed to mix for 1minute, and electroporation was performed for 30 seconds. The parametersfor the poring pulse were: voltage, 2400 V; length, 5 ms; interval, 50ms; number of pulses, 1; polarity, +. The paramters for the transferpulses were: Voltage, 150 V; length, 50 ms; interval, 50 ms; number ofpulses, 20; polarity, +/−. Following electroporation, the cells weretransferred to a recovery module (another growth module), and allowed torecover in SOC medium containing chloramphenicol. Carbenicillin wasadded to the medium after 1 hour, and the cells were allowed to recoverfor another 2 hours. After recovery, the cells were held at 4° C. untilrecovered by the user.

After the automated process and recovery, an aliquot of cells was platedon MacConkey agar base supplemented with lactose (as the sugarsubstrate), chloramphenicol and carbenicillin and grown until coloniesappeared. White colonies represented functionally edited cells, purplecolonies represented un-edited cells. All liquid transfers wereperformed by the automated liquid handling device of the automatedmulti-module cell processing instrument.

The result of the automated processing was that approximately 1.0E⁻⁰³total cells were transformed (comparable to conventional benchtopresults), and the editing efficiency was 83.5%. The lacZ_172 edit in thewhite colonies was confirmed by sequencing of the edited region of thegenome of the cells. Further, steps of the automated cell processingwere observed remotely by webcam and text messages were sent to updatethe status of the automated processing procedure.

Example II: Fully Automated Recursive Editing Run

Recursive editing was successfully achieved using the automatedmulti-module cell processing system. An ampR plasmid backbone and alacZ_V10* editing cassette were assembled via Gibson Assembly® into an“editing vector” in an isothermal nucleic acid assembly module includedin the automated system. Similar to the lacZ_F172 edit, the lacZ_V10edit functionally knocks out the lacZ gene. “lacZ_V10” indicates thatthe edit happens at amino acid position 10 in the lacZ amino acidsequence. Following assembly, the product was de-salted in theisothermal nucleic acid assembly module using AMPure beads, washed with80% ethanol, and eluted in buffer. The first assembled editing vectorand the recombineering-ready electrocompetent E.Coli cells weretransferred into a transformation module for electroporation. Thetransformation module comprised an ADP-EPC cuvette. The cells andnucleic acids were combined and allowed to mix for 1 minute, andelectroporation was performed for 30 seconds. The parameters for theporing pulse were: voltage, 2400 V; length, 5 ms; interval, 50 ms;number of pulses, 1; polarity, +. The parameters for the transfer pulseswere: Voltage, 150 V; length, 50 ms; interval, 50 ms; number of pulses,20; polarity, +/−. Following electroporation, the cells were transferredto a recovery module (another growth module) allowed to recover in SOCmedium containing chloramphenicol. Carbenicillin was added to the mediumafter 1 hour, and the cells were grown for another 2 hours. The cellswere then transferred to a centrifuge module and a media exchange wasthen performed. Cells were resuspended in TB containing chloramphenicoland carbenicillin where the cells were grown to OD600 of 2.7, thenconcentrated and rendered electrocompetent.

During cell growth, a second editing vector was prepared in theisothermal nucleic acid assembly module. The second editing vectorcomprised a kanamycin resistance gene, and the editing cassettecomprised a galK Y145* edit. If successful, the galK Y145* edit conferson the cells the ability to uptake and metabolize galactose. The editgenerated by the galK Y154* cassette introduces a stop codon at the154th amino acid reside, changing the tyrosine amino acid to a stopcodon. This edit makes the galK gene product non-functional and inhibitsthe cells from being able to metabolize galactose. Following assembly,the second editing vector product was de-salted in the isothermalnucleic acid assembly module using AMPure beads, washed with 80%ethanol, and eluted in buffer. The assembled second editing vector andthe electrocompetent E. Coli cells (that were transformed with andselected for the first editing vector) were transferred into atransformation module for electroporation, using the same parameters asdetailed above. Following electroporation, the cells were transferred toa recovery module (another growth module), allowed to recover in SOCmedium containing carbenicillin. After recovery, the cells were held at4° C. until retrieved, after which an aliquot of cells were plated on LBagar supplemented with chloramphenicol, and kanamycin. To quantify bothlacZ and galK edits, replica patch plates were generated on two mediatypes: 1) MacConkey agar base supplemented with lactose (as the sugarsubstrate), chloramphenicol, and kanamycin, and 2) MacConkey agar basesupplemented with galactose (as the sugar substrate), chloramphenicol,and kanamycin. All liquid transfers were performed by the automatedliquid handling device of the automated multi-module cell processingsystem.

In this recursive editing experiment, 41% of the colonies screened hadboth the lacZ and galK edits, the results of which were comparable tothe double editing efficiencies obtained using a “benchtop” or manualapproach.

Alternative Embodiments of Instrument Architecture

FIGS. 12A and 12B illustrate example alternative embodiments ofautomated multi-module cell editing instruments for performing automatedcell processing, e.g., editing in multiple cells in a single iycle. Theautomated multi-module cell editing instruments, for example, may bedesktop instrument s designed for use within a laboratory environment.The automated multi-module cell editing instruments may incorporate amixture of reusable and disposable elements for performing variousstaged operations in conducting automated genome cleavage and/or editingin cells.

FIG. 12A is a block diagram of a first example instrument 1200 forperforming automated cell processing, e.g., editing in multiple cells ina single cycle according to one embodiment of the disclosure. In someimplementations, the instrument 1200 includes a deck 1202, a reagentsupply receptacle 1204 for introducing DNA sample components to theinstrument 1200, a cell supply receptacle 1206 for introducing cells tothe instrument 1200, and a robot handling system 1208 for movingmaterials between modules (for example, modules 1210 a, 1210 b, 1210 c,1210 d) receptacles (for example, receptacles 1204 1206, 1212, 1222,1224, and 1226), and storage units (e.g., units 1216, 1218, 1228, and1214) of the instrument 1200 to perform the automated cell processing.Upon completion of processing of the cell supply 1206, in someembodiments, cell output 1212 may be transferred by the robot handlingsystem 1208 to a storage unit 1214 for temporary storage and laterretrieval.

The robotic handling system 1208, for example, may include an airdisplacement pump to transfer liquids from the various material sourcesto the various modules 1210 and storage unit 1214. In other embodiments,the robotic handling system 1208 may include a pick and place head totransfer containers of source materials (e.g., tubes) from a supplycartridge (not illustrated, discussed in relation to FIG. 1A) to thevarious modules 1210. In some embodiments, one or more cameras or otheroptical sensors (not shown), confirm proper gantry movement andposition.

In some embodiments, the robotic handling system 1208 uses disposabletransfer tips provided in a transfer tip supply 1216 to transfer sourcematerials, reagent 1204 (e.g., nucleic acid assembly), and cells 1206within the instrument 1200. Used transfer tips 1216, for example, may bediscarded in a solid waste unit 1218. In some implementations, the solidwaste unit 1218 contains a kicker to remove tubes from the pick andplace head of robotic handling system 1208.

In some embodiments, the instrument 1200 includes electroporatorcuvettes with sippers that connect to an air displacement pump. In someimplementations, cells 1206 and reagent 1204 are aspirated into theelectroporation cuvette through a sipper, and the cuvette is moved toone or more modules 1210 of the instrument 1200.

In some implementations, the instrument 1200 is controlled by aprocessing system 1220 such as the processing system 1310 of FIG. 13.The processing system 1220 may be configured to operate the instrument100 based on user input. The processing system 1220 may control thetiming, duration, temperature and other operations of the variousmodules 1210 of the instrument 1200. The processing system 1220 may beconnected to a power source (not shown) for the operation of theinstrument 1200.

In some embodiments, instrument 1200 includes a transformation module1210 c for introduction of, e.g., in the context of editing, nucleicacid(s) into the cells 1206. For example, the robotic handling system1208 may transfer the reagent 1204 and cells 1206 to the transformationmodule 1210 c. The transformation module 1210 may conduct any celltransformation or transfection techniques routinely used by those ofskill in the arts of transfection, transformation and microfluidics.Transformation is intended to include to a variety of art-recognizedtechniques for introducing an exogenous nucleic acid sequence (e.g.,DNA) into a target cell, including those transformation and transfectiontechniques. Such methods include, but are not limited to,electroporation, lipofection, optoporation, injection,microprecipitation, microinjection, liposomes, particle bombardment,sonoporation, laser-induced poration, bead transfection, calciumphosphate or calcium chloride co-precipitation, or DEAE-dextran-mediatedtransfection. Transformation can take place in microfuge tubes, testtubes, cuvettes, multi-well plates, microfibers, or flow instrument s.The processing system 1220 may control temperature and operation of thetransformation module 1210 c. In some implementations, the processingsystem 1270 effects operation of the transformation module 1210 caccording to one or more variable controls set by a user.

In some implementations, the transformation module 1210 c is configuredto prepare cells for vector uptake by increasing cell competence with apretreatment solution, 1222, e.g., a sucrose or glycerol wash.Additionally, hybrid techniques that exploit the capabilities ofmechanical and chemical transfection methods can be used, e.g.,magnetofection, a transfection methodology that combines chemicaltransfection with mechanical methods. In another example, cationiclipids may be deployed in combination with gene guns or electroporators.Suitable materials and methods for transforming or transfecting targetcells can be found, e.g., in Green and Sambrook, Molecular Cloning: ALaboratory Manual, 4th, ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 2014), and other laboratory manuals.

Following transformation, in some implementations, the cells may betransferred to a recovery module 1210 d. In some embodiments, therecovery module 1210 d is a combination recovery and induction ofediting module. In the recovery module 1210 d, the cells may be allowedto recover, express the nucleic acids and, in an inducible nucleasesystem, a nuclease is introduced to the cells, e.g., by means oftemporally-controlled induction such as, in some examples, chemical,light, viral, or temperature induction or the introduction of an inducermolecule 1224 for expression of the nuclease.

Following editing, in some implementations, the cells are transferred tothe storage unit 1214, where the cells can be stored as cell output 1212until the cells are removed for further study or retrieval of an editedcell population, e.g., an edited cell library.

In some implementations the instrument 1200 is designed for recursivegenome editing, where multiple edits are sequentially introduced intogenomes inside the cells of a cell population. In some implementations,the reagent supply 1204 is replenished prior to accessing cell output1212 from the storage unit for recursive processing. In otherimplementations, multiple reagent supplies 1204 and/or large volumesthereof may be introduced into the instrument 1200 such that userinteraction is not necessarily required prior to a subsequent processingcycle.

A portion of a cell output 1212 a, in some embodiments, is transferredto an automated cell growth module 1210 a. For example, all of the celloutput 1212 a may be transferred, or a only an aliquot may betransferred such that the instrument retains incrementally modifiedsamples. The cell growth module 1210 a, in some implementations,measures the OD of the cells during growth to ensure they are at adesired concentration prior to induction of editing. Other measures ofcell density and physiological state that can be used include but arenot limited to, pH, dissolved oxygen, released enzymes, acousticproperties, and electrical properties.

To reduce the background of cells that have not received a genome edit,in some embodiments, the growth module 1210 a performs a selectionprocess to enrich for the edited cells using a selective growth medium1226. For example, the introduced nucleic acid can include a gene thatconfers antibiotic resistance or another selectable marker. In someimplementations, multiple selective genes or markers 1226 may beintroduced into the cells during recursive editing. For example,alternating the introduction of selectable markers for sequential roundsof editing can eliminate the background of unedited cells and allowmultiple cycles of the instrument 1200 to select for cells havingsequential genome edits. Suitable antibiotic resistance genes include,but are not limited to, genes such as ampicillin-resistance gene,tetracycline-resistance gene, kanamycin-resistance gene,neomycin-resistance gene, canavanine-resistance gene,blasticidin-resistance gene, hygromycin-resistance gene,puromycin-resistance gene, nd chloramphenicol-resi stance gene.

From the growth module 1210 a, the cells may be transferred to afiltration module 110 b. The filtration module 1210 b or, alternatively,a cell wash and concentration module, may enable media exchange. In someembodiments, removing dead cell background is aided using lyticenhancers such as detergents, osmotic stress, temperature, enzymes,proteases, bacteriophage, reducing agents, or chaotropes. In otherembodiments, cell removal and/or media exchange is used to reduce deadcell background. Waste product from the filtration module 1210 b, insome embodiments, is collected in a liquid waste unit 1228.

After filtration, the cells may be presented to the transformationmodule 1210 c, and then to the recovery module 1210 d and finally to thestorage unit 1214 as detailed above.

Turning to FIG. 12B, similar to FIG. 12A, a second example instrument1240 for performing automated genome cleavage and/or editing in multiplecells in a single cycle includes the deck 1202, the reagent supplyreceptacle 1204 for introducing one or more nucleic acid components tothe instrument 1240, the cell supply receptacle 1206 for introducingcells to the instrument 1240, and the robot handling system 1208 formoving materials between modules (for example, modules 1210 a, 1210 b,1210 c, 1210 f 1210 g, 1210 m, and 1210 h), receptacles (for example,receptacles 1204 1206, 1212, 1214, 1224, 1242, 1244, and 1246), andstorage units (e.g., units 1214, 1216, 1218, and 1228) of the instrument1240 to perform the automated cell processing. Upon completion ofprocessing of the cell supply 1206, in some embodiments, cell output1212 may be transferred by the robot handling system 1208 to the storageunit 1214 for temporary storage and later retrieval.

In some embodiments, the robotic handling system 1208 uses disposabletransfer tips provided in the transfer tip supply 1216 to transfersource materials, a vector backbone 1242, editing oligos 1244, reagenst1204 (e.g., for nucleic acid assembly, nucleic acid purification, torender cells electrocompetent, etc.), and cells 1206 within theinstrument 1240, as described in relation to FIG. 12A.

In other embodiments, the instrument 1240 includes electroporatorcuvettes with sippers that connect to an air displacement pump. In someimplementations, the cells 1206 and the reagent 1204 are aspirated intothe electroporation cuvette through a sipper, and the cuvette is movedto one or more modules 1210 of the instrument 1240.

As described in relation to FIG. 12A, in some implementations, theinstrument 1240 is controlled by the processing system 1220 such as theprocessing system 1310 of FIG. 13.

The instrument 1240, in some embodiments, includes a nucleic acidassembly module 1210 g, and in certain example automated multi-modulecell processing instruments, the nucleic acid assembly module 1210 g mayinclude in some embodiments an isothermal nucleic acid assembly. Asdescribed above, the isothermal nucleic acid assembly module isconfigured to perform the Gibson Assembly® molecular cloning method.

In some embodiments, after assembly of the nucleic acids, the nucleicacids (e.g., in the example of an isothermal nucleic acid assembly, theisothermal nucleic acid assembly mix (nucleic acids+isothermal nucleicacid assembly reagents) are transferred to a purification module 1210 h.Here, unwanted components of the nucleic acid assembly mixture areremoved (e.g., salts, minerals) and, in certain embodiments, theassembled nucleic acids are concentrated. For example, in anillustrative embodiment, in the purification module 1210 h, theisothermal nucleic acid assembly mix may be combined with a no-saltbuffer and magnetic beads, such as Solid Phase Reversible Immobilization(SPRI) magnetic beads or AMPure beads. The isothermal nucleic acidassembly mix may be incubated for sufficient time (e.g., 30 seconds to10 minutes) for the assembled nucleic acids to bind to the magneticbeads. In some embodiments, the purification module includes a magnetconfigured to engage the magnetic beads. The magnet may be engaged sothat the supernatant may be removed from the bound assembled nucleicacids and so that the bound assembled nucleic acids can be washed with,e.g., 80% ethanol. Again, the magnet may be engaged and the 80% ethanolwash solution removed. The magnetic bead/assembled nucleic acids may beallowed to dry, then the assembled nucleic acids may be eluted and themagnet may again be engaged, this time to sequester the beads and toremove the supernatant that contains the eluted assembled nucleic acids.The assembled nucleic acids may then be transferred to thetransformation module (e.g., electroporator in a preferred embodiment).The transformation module may already contain the electrocompetent cellsupon transfer.

In some embodiments, instrument 1240 includes the transformation module1210 c for introduction of the nucleic acid(s) into the cells 1206, asdescribed in relation to FIG. 12A. However, in this circumstance, theassembled nucleic acids 1204, output from the purification module 1210h, are transferred to the transformation module 1210 c for combinationwith the cells 1206.

Following transformation in the transformation module 1210 c, in someimplementations, the cells may be transferred to a recovery module 1210m. In the recovery module 1210 e, the cells may be allowed to recover,express the nucleic acids, and, in an inducible nuclease system, thenuclease is induced, e.g., by means of temporally-controlled inductionsuch as, in some examples, chemical, light, viral, or temperatureinduction or the introduction of the inducer molecule for expression ofthe nuclease.

Following recovery, in some implementations, the cells are transferredto an editing module 1210 f. The editing module 1210 f suppliesappropriate conditions to induce editing of the cells' genomes, e.g.,through expression of the introduced nucleic acids and the induction ofan inducible nuclease. The cells may include an inducible nuclease. Thenuclease may be, in some examples, chemically induced, biologicallyinduced (e.g., via inducible promoter) virally induced, light induced,temperature induced, and/or heat induced within the editing module 1210f.

Following editing, in some implementations, the cells are transferred tothe storage unit 1214 as described in relation to FIG. 12A.

In some implementations, the instrument 1240 is designed for recursivegenome editing, where multiple edits are sequentially introduced intogenomes inside the cells of a cell population. In some implementations,the reagent supply 1204 is replenished prior to accessing cell output1212 from the storage unit for recursive processing. For example,additional vector backbone 1242 and/or editing oligos 1244 may beintroduced into the instrument 1240 for assembly and preparation via thenucleic acid assembly module 1210 g and the purification module 1210 h.In other implementations, multiple vector backbone volumes 1242 and/orediting oligos 1244 may be introduced into the instrument 140 such thatuser interaction is not necessarily required prior to a subsequentprocessing cycle. For each subsequent cycle, the vector backbone 1242and/or editing oligos 1244 may change. Upon preparation of the nucleicacid assembly, the nucleic acid assembly may be provided in the reagentsupply 1204 or another storage region.

A portion of a cell output 1212 a, in some embodiments, is transferredto the automated cell growth module 1210 a, as discussed in relation toFIG. 12A.

To reduce background of cells that have not received a genome edit, insome embodiments, the growth module 1210 a performs a selection processto enrich for the edited cells using a selective growth medium 1226, asdiscussed in relation to FIG. 12A.

From the growth module 1210 a, the cells may be transferred to thefiltration module 1210 b, as discussed in relation to FIG. 12A. Asillustrated, eluant from an eluting supply 1246 (e.g. buffer, glycerol)may be transferred into the filtration module 1210 b for media exchange.

After filtration, the cells may be presented to the transformationmodule 1210 c for transformation, and then to the recovery module 110 mand the editing module 1210 f and finally to the storage unit 1214 asdetailed above.

In some embodiments, the automated multi-module cell processinginstruments of FIG. 12A and/or 12B contain one or more replaceablesupply cartridges and a robotic handling system, as discussed inrelation to FIGS. 1A and 1B. Each cartridge may contain one or more of anucleic acid assembly mix, oligonucleotides, vector, growth media,selection agent (e.g., antibiotics), inducing agent, nucleic acidpurification reagents such as Solid Phase Reversible Immobilization(SPRI) beads, ethanol, and 10% glycerol.

Although the example instruments 1200, 1240 are illustrated as includinga particular arrangement of modules 1210, these arrangements are forillustrative purposes only. For example, in other embodiments, more orfewer modules 1210 may be included within each of the instruments 1200,1240. Also, different modules may be included in the instrument, suchas, e.g., a module that facilitates cell fusion for providing, e.g.,hybridomas, a module that amplifies nucleic acids before assembly,and/or a module that facilitates protein expression and/or secretion.Further, certain modules 1210 may be replicated within certainembodiments, such as the duplicate cell growth modules 110 a, 110 b ofFIG. 1A. Each of the instruments 1200 and 1240, in another example, maybe designed to accept a media cartridge such as the cartridges 104 and106 of FIG. 1A. Further modifications are possible.

Control System for an Automated Multi-Module Cell Processing Instrument

Turning to FIG. 11, a screen shot illustrates an example graphical userinterface (GUI) 1100 for interfacing with an automated multi-module cellprocessing instrument. The interface, for example, may be presented onthe display 236 of FIGS. 1C and 2D. In one example, the GUI 1100 may bepresented by the processing system 1310 of FIG. 13 on the touch screen1316.

In some implementations, the GUI 1100 is divided into a number ofinformation and data entry panes, such as a protocol pane 1102, atemperature pane 1106, an electroporation pane 1108, and a cell growthpane 1110. Further panes are possible. For example, in some embodimentsthe GUI 1100 includes a pane for each module, such as, in some examples,one or more of each of a nucleic acid assembly module, a purificationmodule, a cell growth module, a filtration module, a transformationmodule, an editing module, and a recovery module. The lower panes of theGUI 1100, in some embodiments, represent modules applicable to thepresent work flow (e.g, as selected in the protocol pane 1102 or asdesignated within a script loaded through a script interface (notillustrated)). In some embodiments, a scroll or paging feature may allowthe user to access additional panes not illustrated within the screenshot of FIG. 11.

The GUI 1100, in some embodiments, includes a series of controls 1120for accessing various screens such as the illustrated screen shot (e.g.,through using a home control 1120 a). For example, through selecting anediting control 1120 b, the user may be provided the option to provideone, two or a series of cell processing steps. Through selecting ascript control 1120 c, the user may be provided the opportunity to add anew processing script or alter an existing processing script. The userin some embodiments, may select a help control 1120 d to obtain furtherinformation regarding the features of the GUI 1100 and the automatedmulti-module cell processing instrument. In some implementations, theuser selects a settings control 1120 e to access settings options fordesired processes and/or the GUI 1100 such as, in some examples, timezone, language, units, network access options. A power control 1120 f,when selected, allows the user to power down the automated multi-modulecell processing instrument.

Turning to the protocol pane 1102, in some implementations, a userselects a protocol (e.g., script or work flow) for execution by theautomated multi-module cell processing instrument by entering theprotocol in a protocol entry field 1112 (or, alternatively, drop-downmenu). In other embodiments, the protocol may be selected through aseparate user interface screen, accessed for example by selecting thescript control 1120 b. In another example, the automated multi-modulecell processing instrument may select the protocol and present it in theprotocol entry field 1112. For example, a processing system of theautomated multi-module cell processing instrument may scanmachine-readable indicia positioned on one or more cartridges loadedinto the automated multi-module cell processing instrument to determinethe appropriate protocol. As illustrated, the “Microbe_Kit1 (1.0.2)”protocol has been selected, which may correspond to a kit of cartridgesand other disposable supplies purchased for use with the automatedmulti-module cell processing instrument.

In some implementations, the protocol pane 1102 further includes a startcontrol 1114 a and a stop control 1114 b to control execution of theprotocol presented in the protocol entry field 1112. The GUI 1100 may beprovided on a touch screen interface, for example, where touch selectionof the start control 1114 a starts cell processing, and selection of thestop control 1114 b stops cell processing.

Turning to the run status pane 1104, in some implementations, a chart1116 illustrates stages of the processing of the protocol identified inthe protocol pane 1102. For example, a portion of run completion 1118 ais illustrated in blue, while a portion of current stage 1118 b isillustrated in green, and any errors 1118 c are flagged with markersextending from the point in time along the course of the portion of therun completion 1118 a where the error occurred. A message region 1118 dpresents a percentage of run completed, a percentage of stage completed,and a total number of errors. In some embodiments, upon selection of thechart 1116, the user may be presented with greater details regarding therun status such as, in some examples, identification of the type oferror, a name of the current processing stage (e.g., nucleic acidassembly, purification, cell growth, filtration, transformation,recovery, editing, etc.), and a listing of processing stages within therun. Further, in some embodiments, a run completion time messageindicates a date and time at which the run is estimated to complete. Therun, in some examples, may be indicative of a single cell editingprocess or a series of recursive cell editing processes scheduled forexecution without user intervention. In some embodiments (not shown),the run status pane 1104 additionally illustrates an estimated time atwhich user intervention will be required (e.g., cartridge replacement,solid waste disposal, liquid waste disposal, etc.).

In some implementations, the run status pane 1104 includes a pausecontrol 1124 for pausing cell processing. The user may select to pausethe current run, for example, to correct for an identified error or toconduct manual intervention such as waste removal.

The temperature pane 1106, in some embodiments, illustrates a series oficons 1126 with corresponding messages 1128 indicating temperaturesettings for various apparatus of the automated multi-module cellprocessing instrument. The icons, from left to right, may represent atransformation module 1126 a (e.g., flow-through electroporationcartridge associated with the reagent cartridge 110 c of FIG. 1A or theflow-through electroporation devices 534 of FIG. 5B), a purificationmodule 1126 b, a first growth module 1126 c, a second growth module 1126d, and a filtration module 1126 e. The corresponding messages 1128 a-eidentify a present temperature, low temperature, and high temperature ofthe corresponding module (e.g., for this stage or this run). Inselecting one of the icons 1126, in some embodiments, a graphic displayof temperature of time may be reviewed.

Beneath the temperature pane, in some implementations, a series of panesidentify present status of a number of modules. For example, theelectroporation pane 1108 represents status of a transformation module,while the cell growth pane 1110 represents the status of a growthmodule. In some embodiments, the panes presented here identify status ofa presently operational module (e.g., the module involved in cellprocessing in the current stage) as well as the status of any moduleswhich have already been utilized during the current run (as illustrated,for example, in the run status pane 1104). Past status information, forexample, may present to the user information regarding the parametersused in the prior stage(s) of cell processing.

Turning to the electroporation pane 1108, in some implementations,operational parameters 1130 a of volts, milliamps, and joules arepresented. Additionally, a status message 1132 a may identify additionalinformation regarding the functioning of the transformation module suchas, in some examples, an error status, a time remaining for processing,or contents of the module (e.g., materials added to the module). In someimplementations, an icon 1134 a above the status message 1132 a will bepresented in an active mode (e.g., colorful, “lit up”, in bold, etc.)when the corresponding module is actively processing. Selection of theicon 1134 a, in some embodiments, causes presentation of a graphicdisplay of detailed information regarding the operational parameters1130 a.

Turning to the cell growth pane 1110, in some implementations,operational parameters 1130 b of OD and hours of growth are presented.Additionally, a status message 1132 b may identify additionalinformation regarding the functioning of the growth module such as, insome examples, an error status, a time remaining for processing, orcontents of the module (e.g., materials added to the module). In someimplementations, an icon 1134 b above the status message 1132 b will bepresented in an active mode (e.g., colorful, “lit up”, in bold, etc.)when the corresponding module is actively processing. Selection of theicon 1134 b, in some embodiments, causes presentation of a graphicdisplay of detailed information regarding the operational parameters1130 b.

Next, a hardware description of an example processing system andprocessing environment according to exemplary embodiments is describedwith reference to FIG. 13. In FIG. 13, the processing system 1310includes a CPU 1308 which performs a portion of the processes describedabove. For example, the CPU 1308 may manage the processing stages of themethod 900 of FIG. 9 and/or the workflows of FIGS. 10A-C. The processdata and, scripts, instructions, and/or user settings may be stored inmemory 1302. These process data and, scripts, instructions, and/or usersettings may also be stored on a storage medium disk 1304 such as aportable storage medium (e.g., USB drive, optical disk drive, etc.) ormay be stored remotely. For example, the process data and, scripts,instructions, and/or user settings may be stored in a locationaccessible to the processing system 1310 via a network 1328. Further,the claimed advancements are not limited by the form of thecomputer-readable media on which the instructions of the inventiveprocess are stored. For example, the instructions may be stored in FLASHmemory, RAM, ROM, or any other information processing device with whichthe processing system 1310 communicates, such as a server, computer,smart phone, or other hand-held computing device.

Further, components of the claimed advancements may be provided as autility application, background daemon, or component of an operatingsystem, or combination thereof, executing in conjunction with CPU 1308and an operating system such as with other computing systems known tothose skilled in the art.

CPU 1308 may be an ARM processor, system-on-a-chip (SOC),microprocessor, microcontroller, digital signal processor (DSP), or maybe other processor types that would be recognized by one of ordinaryskill in the art. Further, CPU 1308 may be implemented as multipleprocessors cooperatively working in parallel to perform the instructionsof the inventive processes described above.

The processing system 1310 is part of a processing environment 1300. Theprocessing system 1310 in FIG. 13 also includes a network controller1306 for interfacing with the network 1328 to access additional elementswithin the processing environment 1300. As can be appreciated, thenetwork 1328 can be a public network, such as the Internet, or a privatenetwork such as an LAN or WAN network, or any combination thereof andcan also include PSTN or ISDN sub-networks. The network 1328 can bewireless such as a cellular network including EDGE, 3G and 4G wirelesscellular systems. The wireless network can also be Wi-Fi, Bluetooth, orany other wireless form of communication that is known.

The processing system 1310 further includes a general purpose I/Ointerface 1312 interfacing with a user interface (e.g., touch screen)1316, one or more sensors 1314, and one or more peripheral devices 1318.The peripheral I/O devices 1318 may include, in some examples, a videorecording system, an audio recording system, microphone, externalstorage devices, and/or external speaker systems. The one or moresensors 1314 may include one or more of a gyroscope, an accelerometer, agravity sensor, a linear accelerometer, a global positioning system, abar code scanner, a QR code scanner, an RFID scanner, a temperaturemonitor, and a lighting system or lighting element.

The general purpose storage controller 1324 connects the storage mediumdisk 1304 with communication bus 1340, such as a parallel bus or aserial bus such as a Universal Serial Bus (USB), or similar, forinterconnecting all of the components of the processing system. Adescription of the general features and functionality of the storagecontroller 1324, network controller 1306, and general purpose I/Ointerface 1312 is omitted herein for brevity as these features areknown.

The processing system 1310, in some embodiments, includes one or moreonboard and/or peripheral sensors 1314. The sensors 1314, for example,can be incorporated directly into the internal electronics and/or ahousing of the automated multi-module processing instrument. A portionof the sensors 1314 can be in direct physical contact with the I/Ointerface 1312, e.g., via a wire; or in wireless contact e.g., via aBluetooth, Wi-Fi or NFC connection. For example, a wirelesscommunications controller 1326 may enable communications between one ormore wireless sensors 1314 and the I/O interface 1312. Furthermore, oneor more sensors 1314 may be in indirect contact e.g., via intermediaryservers or storage devices that are based in the network 1328; or in(wired, wireless or indirect) contact with a signal accumulatorsomewhere within the automated multi-module cell processing instrument,which in turn is in (wired or wireless or indirect) contact with the I/Ointerface 1312.

A group of sensors 1314 communicating with the I/O interface 1312 may beused in combination to gather a given signal type from multiple placesin order to generate a more complete map of signals. One or more sensors1314 communicating with the I/O interface 1312 can be used as acomparator or verification element, for example to filter, cancel, orreject other signals.

In some embodiments, the processing environment 1300 includes acomputing device 1338 communicating with the processing system 1310 viathe wireless communications controller 1326. For example, the wirelesscommunications controller 1326 may enable the exchange of emailmessages, text messages, and/or software application alerts designatedto a smart phone or other personal computing device of a user.

The processing environment 1300, in some implementations, includes arobotic material handling system 1322. The processing system 1310 mayinclude a robotics controller 1320 for issuing control signals toactuate elements of the robotic material handling system, such asmanipulating a position of a gantry, lowering or raising a sipper orpipettor element, and/or actuating pumps and valves to cause liquidtransfer between a sipper/pipettor and various vessels (e.g., chambers,vials, etc.) in the automated multi-module cell processing instrument.The robotics controller 1320, in some examples, may include a hardwaredriver, firmware element, and/or one or more algorithms or softwarepackages for interfacing the processing system 1310 with the roboticsmaterial handling system 1322.

In some implementations, the processing environment 1310 includes one ormore module interfaces 1332, such as, in some examples, one or moresensor interfaces, power control interfaces, valve and pump interfaces,and/or actuator interfaces for activating and controlling processing ofeach module of the automated multi-module processing system. Forexample, the module interfaces 1332 may include an actuator interfacefor the drive motor 864 of rotating cell growth device 850 (FIG. 8D) anda sensor interface for the detector board 872 that senses opticaldensity of cell growth within rotating growth vial 800. A modulecontroller 1330, in some embodiments, is configured to interface withthe module interfaces 1332. The module controller 1330 may include oneor many controllers (e.g., possibly one controller per module, althoughsome modules may share a single controller). The module controller 1330,in some examples, may include a hardware driver, firmware element,and/or one or more algorithms or software packages for interfacing theprocessing system 1310 with the module interfaces 1332.

The processing environment 1310, in some implementations, includes athermal management system 1336 for controlling climate conditions withinthe housing of the automated multi-module processing system. The thermalmanagement system 1336 may additional control climate conditions withinone or more modules of the automated multi-module cell processinginstrument. The processing system 1310, in some embodiments, includes atemperature controller 1334 for interfacing with the thermal managementsystem 1336. The temperature controller 1334, in some examples, mayinclude a hardware driver, firmware element, and/or one or morealgorithms or software packages for interfacing the processing system1310 with the thermal management system 1336.

Production of Cell Libraries Using Automated Editing Methods, Modules,Instruments and Systems

In one aspect, the present disclosure provides automated editingmethods, modules, instruments, and automated multi-module cell editinginstruments for creating a library of cells that vary the expression,levels and/or activity of RNAs and/or proteins of interest in variouscell types using various editing strategies, as described herein in moredetail. Accordingly, the disclosure is intended to cover edited celllibraries created by the automated editing methods, automatedmulti-module cell editing instruments of the disclosure. These celllibraries may have different targeted edits, including but not limitedto gene knockouts, gene knock-ins, insertions, deletions, singlenucleotide edits, short tandem repeat edits, frameshifts, triplet codonexpansion, and the like in cells of various organisms. These edits canbe directed to coding or non-coding regions of the genome, and arepreferably rationally designed.

In other aspects, the present disclosure provides automated editingmethods, automated multi-module cell editing instruments for creating alibrary of cells that vary DNA-linked processes. For example, the celllibrary may include individual cells having edits in DNA binding sitesto interfere with DNA binding of regulatory elements that modulateexpression of selected genes. In addition, cell libraries may includeedits in genomic DNA that impact on cellular processes such asheterochromatin formation, switch-class recombination and VDJrecombination.

In specific aspects, the cell libraries are created using multiplexedediting of individual cells within a cell population, with multiplecells within a cell population are edited in a single round of editing,i.e., multiple changes within the cells of the cell library are in asingle automated operation. The libraries that can be created in asingle multiplexed automated operation can comprise as many as 500edited cells, 1000 edited cells, 2000 edited cells, 5000 edited cells,10,000 edited cells, 50,000 edited cells, 100,000 edited cells, 200,000edited cells, 300,000 edited cells, 400,000 edited cells, 500,000 editedcells, 600,000 edited cells, 700,000 edited cells, 800,000 edited cells,900,000 edited cells, 1,000,000 edited cells, 2,000,000 edited cells,3,000,000 edited cells, 4,000,000 edited cells, 5,000,000 edited cells,6,000,000 edited cells, 7,000,000 edited cells, 8,000,000 edited cells,9,000,000 edited cells, 10,000,000 edited cells or more.

In other specific aspects, the cell libraries are created usingrecursive editing of individual cells within a cell population, withedits being added to the individual cells in two or more rounds ofediting. The use of recursive editing results in the amalgamation of twoor more edits targeting two or more sites in the genome in individualcells of the library. The libraries that can be created in an automatedrecursive operation can comprise as many as 500 edited cells, 1000edited cells, 2000 edited cells, 5000 edited cells, 10,000 edited cells,50,000 edited cells, 100,000 edited cells, 200,000 edited cells, 300,000edited cells, 400,000 edited cells, 500,000 edited cells, 600,000 editedcells, 700,000 edited cells, 800,000 edited cells, 900,000 edited cells,1,000,000 edited cells, 2,000,000 edited cells, 3,000,000 edited cells,4,000,000 edited cells, 5,000,000 edited cells, 6,000,000 edited cells,7,000,000 edited cells, 8,000,000 edited cells, 9,000,000 edited cells,10,000,000 edited cells or more.

Examples of non-automated editing strategies that can be modified basedon the present specification to utilize the automated systems can befound, e.g., U.S. Pat. Nos. 8,110,360, 8,332,160, 9,988,624,20170316353, and 20120277120.

In specific aspects, recursive editing can be used to first create acell phenotype, and then later rounds of editing used to reverse thephenotype and/or accelerate other cell properties.

In some aspects, the cell library comprises edits for the creation ofunnatural amino acids in a cell.

In specific aspects, the disclosure provides edited cell librarieshaving edits in one or more regulatory elements created using theautomated editing methods, automated multi-module cell editinginstruments of the disclosure. The term “regulatory element” refers tonucleic acid molecules that can influence the transcription and/ortranslation of an operably linked coding sequence in a particularenvironment and/or context. This term is intended to include allelements that promote or regulate transcription, and RNA stabilityincluding promoters, core elements required for basic interaction of RNApolymerase and transcription factors, upstream elements, enhancers, andresponse elements (see, e.g., Lewin, “Genes V” (Oxford University Press,Oxford) pages 847-873). Exemplary regulatory elements in prokaryotesinclude, but are not limited to, promoters, operator sequences and aribosome binding sites. Regulatory elements that are used in eukaryoticcells may include, but are not limited to, promoters, enhancers,insulators, splicing signals and polyadenylation signals.

Preferably, the edited cell library includes rationally designed editsthat are designed based on predictions of protein structure, expressionand/or activity in a particular cell type. For example, rational designmay be based on a system-wide biophysical model of genome editing with aparticular nuclease and gene regulation to predict how different editingparameters including nuclease expression and/or binding, growthconditions, and other experimental conditions collectively control thedynamics of nuclease editing. See, e.g., Farasat and Salis, PLoS ComputBiol., 29:12(1):e1004724 (2016).

In one aspect, the present disclosure provides the creation of a libraryof edited cells with various rationally designed regulatory sequencescreated using the automated editing instrumentation, systems and methodsof the invention. For example, the edited cell library can includeprokaryotic cell populations created using set of constitutive and/orinducible promoters, enhancer sequences, operator sequences and/orribosome binding sites. In another example, the edited cell library caninclude eukaryotic sequences created using a set of constitutive and/orinducible promoters, enhancer sequences, operator sequences, and/ordifferent Kozak sequences for expression of proteins of interest.

In some aspects, the disclosure provides cell libraries including cellswith rationally designed edits comprising one or more classes of editsin sequences of interest across the genome of an organism. In specificaspects, the disclosure provides cell libraries including cells withrationally designed edits comprising one or more classes of edits insequences of interest across a subset of the genome. For example, thecell library may include cells with rationally designed edits comprisingone or more classes of edits in sequences of interest across the exome,e.g., every or most open reading frames of the genome. For example, thecell library may include cells with rationally designed edits comprisingone or more classes of edits in sequences of interest across the kinome.In yet another example, the cell library may include cells withrationally designed edits comprising one or more classes of edits insequences of interest across the secretome. In yet other aspects, thecell library may include cells with rationally designed edits created toanalyze various isoforms of proteins encoded within the exome, and thecell libraries can be designed to control expression of one or morespecific isoforms, e.g., for transcriptome analysis.

Importantly, in certain aspects the cell libraries may comprise editsusing randomized sequences, e.g., randomized promoter sequences, toreduce similarity between expression of one or more proteins inindividual cells within the library. Additionally, the promoters in thecell library can be constitutive, inducible or both to enable strongand/or titratable expression.

In other aspects, the present disclosure provides automated editingmethods, automated multi-module cell editing instruments for creating alibrary of cells comprising edits to identify optimum expression of aselected gene target. For example, production of biochemicals throughmetabolic engineering often requires the expression of pathway enzymes,and the best production yields are not always achieved by the highestamount of the target pathway enzymes in the cell, but rather byfine-tuning of the expression levels of the individual enzymes andrelated regulatory proteins and/or pathways. Similarly, expressionlevels of heterologous proteins sometimes can be experimentally adjustedfor optimal yields.

The most obvious way that transcription impacts on gene expressionlevels is through the rate of Pol II initiation, which can be modulatedby combinations of promoter or enhancer strength and trans-activatingfactors (Kadonaga, et al., Cell, 116(2):247-57 (2004). In eukaryotes,elongation rate may also determine gene expression patterns byinfluencing alternative splicing (Cramer et al., PNAS USA,94(21):11456-60 (1997). Failed termination on a gene can impair theexpression of downstream genes by reducing the accessibility of thepromoter to Pol II (Greger, et al., 2000 PNAS USA, 97(15):8415-20(2000). This process, known as transcriptional interference, isparticularly relevant in lower eukaryotes, as they often have closelyspaced genes.

In some embodiments, the present disclosure provides methods foroptimizing cellular gene transcription. Gene transcription is the resultof several distinct biological phenomena, including transcriptionalinitiation (RNAp recruitment and transcriptional complex formation),elongation (strand synthesis/extension), and transcriptional termination(RNAp detachment and termination).

Site Directed Mutagenesis

Cell libraries can be created using the automated editing methods,modules, instruments and systems employing site-directed mutagenesis,i.e., when the amino acid sequence of a protein or other genomic featuremay be altered by deliberately and precisely by mutating the protein orgenomic feature. These cell lines can be useful for various purposes,e.g., for determining protein function within cells, the identificationof enzymatic active sites within cells, and the design of novelproteins. For example, site-directed mutagenesis can be used in amultiplexed fashion to exchange a single amino acid in the sequence of aprotein for another amino acid with different chemical properties. Thisallows one to determine the effect of a rationally designed or randomlygenerated mutation in individual cells within a cell population. See,e.g., Berg, et al. Biochemistry, Sixth Ed. (New York: W.H. Freeman andCompany) (2007).

In another example, edits can be made to individual cells within a celllibrary to substitute amino acids in binding sites, such as substitutionof one or more amino acids in a protein binding site for interactionwithin a protein complex or substitution of one or more amino acids inenzymatic pockets that can accommodate a cofactor or ligand. This classof edits allows the creation of specific manipulations to a protein tomeasure certain properties of one or more proteins, includinginteraction with other cofactors, ligands, etc. within a proteincomplex.

In yet another examples, various edit types can be made to individualcells within a cell library using site specific mutagenesis for studyingexpression quantitative trait loci (eQTLs). An eQTL is a locus thatexplains a fraction of the genetic variance of a gene expressionphenotype. The libraries of the invention would be useful to evaluateand link eQTLs to actual diseased states.

In specific aspects, the edits introduced into the cell libraries of thedisclosure may be created using rational design based on known orpredicted structures of proteins. See, e.g., Chronopoulou EG and Labrou,Curr Protoc Protein Sci.; Chapter 26:Unit 26.6 (2011). Suchsite-directed mutagenesis can provide individual cells within a librarywith one or more site-directed edits, and preferably two or moresite-directed edits (e.g., combinatorial edits) within a cellpopulation.

In other aspects, cell libraries of the disclosure are created usingsite-directed codon mutation “scanning” of all or substantially all ofthe codons in the coding region of a gene. In this fashion, individualedits of specific codons can be examined for loss-of-function orgain-of-function based on specific polymorphisms in one or more codonsof the gene. These libraries can be a powerful tool for determiningwhich genetic changes are silent or causal of a specific phenotype in acell or cell population. The edits of the codons may be randomlygenerated or may be rationally designed based on known polymorphismsand/or mutations that have been identified in the gene to be analyzed.Moreover, using these techniques on two or more genes in a single in apathway in a cell may determine potential protein:protein interactionsor redundancies in cell functions or pathways.

For example, alanine scanning can be used to determine the contributionof a specific residue to the stability or function of given protein.See, e.g., Lefèvre, et al., Nucleic Acids Research, Volume 25(2):447-448(1997). Alanine is often used in this codon scanning technique becauseof its non-bulky, chemically inert, methyl functional group that canmimic the secondary structure preferences that many of the other aminoacids possess. Codon scanning can also be used to determine whether theside chain of a specific residue plays a significant role in cellfunction and/or activity. Sometimes other amino acids such as valine orleucine can be used in the creation of codon scanning cell libraries ifconservation of the size of mutated residues is needed.

In other specific aspects, cell libraries can be created using theautomated editing methods, automated multi-module cell editinginstruments of the invention to determine the active site of a proteinsuch as an enzyme or hormone, and to elucidate the mechanism of actionof one or more of these proteins in a cell library. Site-directedmutagenesis associated with molecular modeling studies can be used todiscover the active site structure of an enzyme and consequently itsmechanism of action. Analysis of these cell libraries can provide anunderstanding of the role exerted by specific amino acid residues at theactive sites of proteins, in the contacts between subunits of proteincomplexes, on intracellular trafficking and protein stability/half-lifein various genetic backgrounds.

Saturation Mutagenesis

In some aspects, the cell libraries created using the automated editingmethods, automated multi-module cell editing instruments of thedisclosure may saturation mutagenesis libraries, in which a single codonor set of codons is randomized to produce all possible amino acids atthe position of a particular gene or genes of interest. These celllibraries can be particularly useful to generate variants, e.g., fordirected evolution. See, e.g., Chica, et al., Current Opinion inBiotechnology 16 (4): 378-384 (2005); nd Shivange, Current Opinion inChemical Biology, 13 (1): 19-25.

In some aspects, edits comprising different degenerate codons can beused to encode sets of amino acids in the individual cells in thelibraries. Because some amino acids are encoded by more codons thanothers, the exact ratio of amino acids cannot be equal. In certainaspects, more restricted degenerate codons are used. ‘NNK’ and ‘NNS’have the benefit of encoding all 20 amino acids, but still encode a stopcodon 3% of the time. Alternative codons such as ‘NDT’, ‘DBK’ avoid stopcodons entirely, and encode a minimal set of amino acids that stillencompass all the main biophysical types (anionic, cationic, aliphatichydrophobic, aromatic hydrophobic, hydrophilic, small).

In specific aspects, the non-redundant saturation mutagenesis, in whichthe most commonly used codon for a particular organism is used in thesaturation mutagenesis editing process.

Promoter Swaps and Ladders

One mechanism for analyzing and/or optimizing expression of one or moregenes of interest is through the creation of a “promoter swap” celllibrary, in which the cells comprise genetic edits that have specificpromoters linked to one or more genes of interest. Accordingly, the celllibraries created using the methods, automated multi-module cell editinginstruments of the disclosure may be promoter swap cell libraries, whichcan be used, e.g., to increase or decrease expression of a gene ofinterest to optimize a metabolic or genetic pathway. In some aspects,the promoter swap cell library can be used to identify an increase orreduction in the expression of a gene that affects cell vitality orviability, e.g., a gene encoding a protein that impacts on the growthrate or overall health of the cells. In some aspects, the promoter swapcell library can be used to create cells having dependencies and logicbetween the promoters to create synthetic gene networks. In someaspects, the promoter swaps can be used to control cell to cellcommunication between cells of both homogeneous and heterogeneous(complex tissues) populations in nature.

The cell libraries can utilize any given number of promoters that havebeen grouped together based upon exhibition of a range of expressionstrengths and any given number of target genes. The ladder of promotersequences vary expression of at least one locus under at least onecondition. This ladder is then systematically applied to a group ofgenes in the organism using the automated editing methods, automatedmulti-module cell editing instruments of the disclosure.

In specific aspects, the cell library formed using the automated editingprocesses, modules and systems of the disclosure include individualcells that are representative of a given promoter operably linked to oneor more target genes of interest in an otherwise identical geneticbackground. Examples of non-automated editing strategies that can bemodified to utilize the automated systems can be found, e.g., in U.S.Pat. No. 9,988,624.

In specific aspects, the promoter swap cell library is produced byediting a set of target genes to be operably linked to a pre-selectedset of promoters that act as a “promoter ladder” for expression of thegenes of interest. For example, the cells are edited so that one or moreindividual genes of interest are edited to be operably linked with thedifferent promoters in the promoter ladder. When an endogenous promoterdoes not exist, its sequence is unknown, or it has been previouslychanged in some manner, the individual promoters of the promoter laddercan be inserted in front of the genes of interest. These produced celllibraries have individual cells with an individual promoter of theladder operably linked to one or more target genes in an otherwiseidentical genetic context.

The promoters are generally selected to result in variable expressionacross different loci, and may include inducible promoters, constitutivepromoters, or both.

The set of target genes edited using the promoter ladder can include allor most open reading frames (ORFs) in a genome, or a selected subset ofthe genome, e.g., the ORFs of the kinome or a secretome. In someaspects, the target genes can include coding regions for variousisoforms of the genes, and the cell libraries can be designed toexpression of one or more specific isoforms, e.g., for transcriptomeanalysis using various promoters.

The set of target genes can also be genes known or suspected to beinvolved in a particular cellular pathway, e.g. a regulatory pathway orsignaling pathway. The set of target genes can be ORFs related tofunction, by relation to previously demonstrated beneficial edits(previous promoter swaps or previous SNP swaps), by algorithmicselection based on epistatic interactions between previously generatededits, other selection criteria based on hypotheses regarding beneficialORF to target, or through random selection. In specific embodiments, thetarget genes can comprise non-protein coding genes, including non-codingRNAs.

Editing of other functional genetic elements, including insulatorelements and other genomic organization elements, can also be used tosystematically vary the expression level of a set of target genes, andcan be introduced using the methods, automated multi-module cell editinginstruments of the disclosure. In one aspect, a population of cells isedited using a ladder of enhancer sequences, either alone or incombination with selected promoters or a promoter ladder, to create acell library having various edits in these enhancer elements. In anotheraspect, a population of cells is edited using a ladder of ribosomebinding sequences, either alone or in combination with selectedpromoters or a promoter ladder, to create a cell library having variousedits in these ribosome binding sequences.

In another aspect, a population of cells is edited to allow theattachment of various mRNA and/or protein stabilizing or destabilizingsequences to the 5′ or 3′ end, or at any other location, of a transcriptor protein.

In certain aspects, a population of cells of a previously establishedcell line may be edited using the automated editing methods, modules,instruments, and systems of the disclosure to create a cell library toimprove the function, health and/or viability of the cells. For example,many industrial strains currently used for large scale manufacturinghave been developed using random mutagenesis processes iteratively overa period of many years, sometimes decades. Unwanted neutral anddetrimental mutations were introduced into strains along with beneficialchanges, and over time this resulted in strains with deficiencies inoverall robustness and key traits such as growth rates. In anotherexample, mammalian cell lines continue to mutate through the passage ofthe cells over periods of time, and likewise these cell lines can becomeunstable and acquire traits that are undesirable. The automated editingmethods, automated multi-module cell editing instruments of thedisclosure can use editing strategies such as SNP and/or STR swapping,indel creation, or other techniques to remove or change the undesirablegenome sequences and/or introducing new genome sequences to address thedeficiencies while retaining the desirable properties of the cells.

When recursive editing is used, the editing in the individual cells inthe edited cell library can incorporate the inclusion of “landing pads”in an ectopic site in the genome (e.g., a CarT locus) to optimizeexpression, stability and/or control.

In some embodiments, each library produced having individual cellscomprising one or more edits (either introducing or removing) iscultured and analyzed under one or more criteria (e.g., production of achemical or product of interest). The cells possessing the specificcriteria are then associated, or correlated, with one or more particularedits in the cell. In this manner, the effect of a given edit on anynumber of genetic or phenotypic traits of interest can be determined.The identification of multiple edits associated with particular criteriaor enhanced functionality/robustness may lead to cells with highlydesirable characteristics.

Knock-out or Knock-in Libraries

In certain aspects, the present disclosure provides automated editingmethods, modules, instruments and systems for creating a library ofcells having “knock-out” (KO) or “knock-in” (KI) edits of various genesof interest. Thus, the disclosure is intended to cover edited celllibraries created by the automated editing methods, automatedmulti-module cell editing instruments of the disclosure that have one ormore mutations that remove or reduce the expression of selected genes ofinterest to interrogate the effect of these edits on gene function inindividual cells within the cell library.

The cell libraries can be created using targeted gene KO (e.g., viainsertion/deletion) or KOs (e.g., via homologous directed repair). Forexample, double strand breaks are often repaired via the non-homologousend joining DNA repair pathway. The repair is known to be error prone,and thus insertions and deletions may be introduced that can disruptgene function. Preferably the edits are rationally designed tospecifically affect the genes of interest, and individual cells can becreated having a KI or KI of one or more locus of interest. Cells havinga KO or KI of two or more loci of interest can be created usingautomated recursive editing of the disclosure.

In specific aspects, the KO or KI cell libraries are created usingsimultaneous multiplexed editing of cells within a cell population, andmultiple cells within a cell population are edited in a single round ofediting, i.e., multiple changes within the cells of the cell library arein a single automated operation. In other specific aspects, the celllibraries are created using recursive editing of individual cells withina cell population, and results in the amalgamation of multiple edits oftwo or more sites in the genome into single cells.

SNP or Short Tandem Repeat Swaps

In one aspect, cell libraries are created using the automated editingmethods, automated multi-module cell editing instruments of thedisclosure by systematic introducing or substituting single nucleotidepolymorphisms (“SNPs”) into the genomes of the individual cells tocreate a “SNP swap” cell library. In some embodiments, the SNP swappingmethods of the present disclosure include both the addition ofbeneficial SNPs, and removing detrimental and/or neutral SNPs. The SNPswaps may target coding sequences, non-coding sequences, or both.

In another aspect, a cell library is created using the automated editingmethods, modules, instruments, instruments, and systems of thedisclosure by systematic introducing or substituting short tandemrepeats (“STR”) into the genomes of the individual cells to create an“STR swap” cell library. In some embodiments, the STR swapping methodsof the present disclosure include both the addition of beneficial STRs,and removing detrimental and/or neutral STRs. The STR swaps may targetcoding sequences, non-coding sequences, or both.

In some embodiments, the SNP and/or STR swapping used to create the celllibrary is multiplexed, and multiple cells within a cell population areedited in a single round of editing, i.e., multiple changes within thecells of the cell library are in a single automated operation. In otherembodiments, the SNP and/or STR swapping used to create the cell libraryis recursive, and results in the amalgamation of multiple beneficialsequences and/or the removal of detrimental sequences into single cells.Multiple changes can be either a specific set of defined changes or apartly randomized, combinatorial library of mutations. Removal ofdetrimental mutations and consolidation of beneficial mutations canprovide immediate improvements in various cellular processes. Removal ofgenetic burden or consolidation of beneficial changes into a strain withno genetic burden also provides a new, robust starting point foradditional random mutagenesis that may enable further improvements.

SNP swapping overcomes fundamental limitations of random mutagenesisapproaches as it is not a random approach, but rather the systematicintroduction or removal of individual mutations across cells.

Splice Site Editing

RNA splicing is the process during which introns are excised and exonsare spliced together to create the mRNA that is translated into aprotein. The precise recognition of splicing signals by cellularmachinery is critical to this process. Accordingly, in some aspects, apopulation of cells is edited using a systematic editing to known and/orpredicted splice donor and/or acceptor sites in various loci to create alibrary of splice site variants of various genes. Such editing can helpto elucidate the biological relevance of various isoforms of genes in acellular context. Sequences for rational design of splicing sites ofvarious coding regions, including actual or predicted mutationsassociated with various mammalian disorders, can be predicted usinganalysis techniques such as those found in Nalla and Rogan, Hum Mutat,25:334-342 (2005); Divina, et al., Eur J Hum Genet, 17:759-765 (2009);Desmet, et el., Nucleic Acids Res, 37:e67 (2009); Faber, et al., BMCBioinformatics, 12(suppl 4): S2 (2011).

Start/Stop Codon Exchanges and Incorporation of Nucleic Acid Analogs

In some aspects, the present disclosure provides for the creation ofcell libraries using the automated editing methods, modules, instrumentsand systems of the disclosure, where the libraries are created byswapping start and stop codon variants throughout the genome of anorganism or for a selected subset of coding regions in the genome, e.g.,the kinome or secretome. In the cell library, individual cells will haveone or more start or stop codons replacing the native start or stopcodon for one or more gene of interest.

For example, typical start codons used by eukaryotes are ATG (AUG) andprokaryotes use ATG (AUG) the most, followed by GTG (GUG) and TTG (UUG).The cell library may include individual cells having substitutions forthe native start codons for one or more genes of interest.

In some aspects, the present disclosure provides for automated creationof a cell library by replacing ATG start codons with TTG in front ofselected genes of interest. In other aspects, the present disclosureprovides for automated creation of a cell library by replacing ATG startcodons with GTG. In other aspects, the present disclosure provides forautomated creation of a cell library by replacing GTG start codons withATG. In other aspects, the present disclosure provides for automatedcreation of a cell library by replacing GTG start codons with TTG. Inother aspects, the present disclosure provides for automated creation ofa cell library by replacing TTG start codons with ATG. In other aspects,the present disclosure provides for automated creation of a cell libraryby replacing TTG start codons with GTG.

In other examples, typical stop codons for S. cerevisiae and mammals areTAA (UAA) and TGA (UGA), respectively. The typical stop codon formonocotyledonous plants is TGA (UGA), whereas insects and E. colicommonly use TAA (UAA) as the stop codon (Dalphin. et al., Nucl. AcidsRes., 24: 216-218 (1996)). The cell library may include individual cellshaving substitutions for the native stop codons for one or more genes ofinterest.

In some aspects, the present disclosure provides for automated creationof a cell library by replacing TAA stop codons with TAG. In otheraspects, the present disclosure provides for automated creation of acell library by replacing TAA stop codons with TGA. In other aspects,the present disclosure provides for automated creation of a cell libraryby replacing TGA stop codons with TAA. In other aspects, the presentdisclosure provides for automated creation of a cell library byreplacing TGA stop codons with TAG. In other aspects, the presentdisclosure provides for automated creation of a cell library byreplacing TAG stop codons with TAA. In other aspects, the presentinvention teaches automated creation of a cell library by replacing TAGstop codons with TGA.

Terminator Swaps and Ladders

One mechanism for identifying optimum termination of a pre-spliced mRNAof one or more genes of interest is through the creation of a“terminator swap” cell library, in which the cells comprise geneticedits that have specific terminator sequences linked to one or moregenes of interest. Accordingly, the cell libraries created using themethods, modules, instruments and systems of the disclosure may beterminator swap cell libraries, which can be used, e.g., to affect mRNAstability by releasing transcripts from sites of synthesis. In otherembodiments, the terminator swap cell library can be used to identify anincrease or reduction in the efficiency of transcriptional terminationand thus accumulation of unspliced pre-mRNA (e.g., West and Proudfoot,Mol Cell.; 33(3-9); 354-364 (2009) and/or 3′ end processing (e.g., West,et al., Mol Cell. 29(5):600-10 (2008)). In the case where a gene islinked to multiple termination sites, the edits may edit a combinationof edits to multiple terminators that are associated with a gene.Additional amino acids may also be added to the ends of proteins todetermine the effect on the protein length on terminators.

The cell libraries can utilize any given number of edits of terminatorsthat have been selected for the terminator ladder based upon exhibitionof a range of activity and any given number of target genes. The ladderof terminator sequences vary expression of at least one locus under atleast one condition. This ladder is then systematically applied to agroup of genes in the organism using the automated editing methods,modules, instruments and systems of the disclosure.

In some aspects, the present disclosure provides for the creation ofcell libraries using the automated editing methods, modules, instrumentsand systems of disclosure, where the libraries are created to editterminator signals in one or more regions in the genome in theindividual cells of the library. Transcriptional termination ineukaryotes operates through terminator signals that are recognized byprotein factors associated with the RNA polymerase II. For example, thecell library may contain individual eukaryotic cells with edits in genesencoding polyadenylation specificity factor (CPSF) and cleavagestimulation factor (CstF) and or gene encoding proteins recruited byCPSF and CstF factors to termination sites. In prokaryotes, twoprincipal mechanisms, termed Rho-independent and Rho-dependenttermination, mediate transcriptional termination. For example, the celllibrary may contain individual prokaryotic cells with edits in genesencoding proteins that affect the binding, efficiency and/or activity ofthese termination pathways.

In certain aspects, the present disclosure provides methods of selectingtermination sequences (“terminators”) with optimal properties. Forexample, in some embodiments, the present disclosure teaches providesmethods for introducing and/or editing one or more terminators and/orgenerating variants of one or more terminators within a host cell, whichexhibit a range of activity. A particular combination of terminators canbe grouped together as a terminator ladder, and cell libraries of thedisclosure include individual cells that are representative ofterminators operably linked to one or more target genes of interest inan otherwise identical genetic background. Examples of non-automatedediting strategies that can be modified to utilize the automatedinstruments can be found, e.g., in U.S. Pat. No. 9,988,624 to Serber etal., entitled “Microbial strain improvement by a HTP genomic engineeringplatform.”

In specific aspects, the terminator swap cell library is produced byediting a set of target genes to be operably linked to a pre-selectedset of terminators that act as a “terminator ladder” for expression ofthe genes of interest. For example, the cells are edited so that theendogenous promoter is operably linked to the individual genes ofinterest are edited with the different promoters in the promoter ladder.When the endogenous promoter does not exist, its sequence is unknown, orit has been previously changed in some manner, the individual promotersof the promoter ladder can be inserted in front of the genes ofinterest. These produced cell libraries have individual cells with anindividual promoter of the ladder operably linked to one or more targetgenes in an otherwise identical genetic context. The terminator ladderin question is then associated with a given gene of interest.

The terminator ladder can be used to more generally affect terminationof all or most ORFs in a genome, or a selected subset of the genome,e.g., the ORFs of a kinome or a secretome. The set of target genes canalso be genes known or suspected to be involved in a particular cellularpathway, e.g. a regulatory pathway or signaling pathway. The set oftarget genes can be ORFs related to function, by relation to previouslydemonstrated beneficial edits (previous promoter swaps or previous SNPswaps), by algorithmic selection based on epistatic interactions betweenpreviously generated edits, other selection criteria based on hypothesesregarding beneficial ORF to target, or through random selection. Inspecific embodiments, the target genes can comprise non-protein codinggenes, including non-coding RNAs.

While certain embodiments have been described, these embodiments havebeen presented by way of example only, and are not intended to limit thescope of the present disclosures. Indeed, the novel methods,apparatuses, modules, instruments and systems described herein can beembodied in a variety of other forms; furthermore, various omissions,substitutions and changes in the form of the methods, apparatuses,modules, instruments and systems described herein can be made withoutdeparting from the spirit of the present disclosures. The accompanyingclaims and their equivalents are intended to cover such forms ormodifications as would fall within the scope and spirit of the presentdisclosures.

The invention claimed is:
 1. An automated stand-alone multi-module cellediting instrument comprising: a housing configured to house all or someof the modules; a receptacle configured to receive cells; one or morereceptacles configured to receive nucleic acids; a growth module forgrowing cells for transformation; a transformation module configured tointroduce the nucleic acids into the cells; a nuclease-directed editingmodule configured to allow the introduced nucleic acids to edit nucleicacids in the cells; a processor configured to operate the automatedmulti-module cell editing instrument based on user input and/orselection of a pre-programmed script; and an automated liquid handlingsystem to move liquids from the cell receptacle to the growth module,from the growth module to the transformation module and from thetransformation module to the nuclease-directed editing module withoutuser intervention.
 2. The automated stand-alone multi-module cellediting instrument of claim 1, wherein the nucleic acids in the one ormore receptacles comprise a backbone and an editing cassette, and theautomated multi-module cell editing instrument further comprises anucleic acid assembly module.
 3. The automated stand-alone multi-modulecell editing instrument of claim 1, wherein the automated liquidhandling system comprises a sipper or pipettor.
 4. The automatedstand-alone multi-module cell editing instrument of claim 2, wherein thenucleic acid assembly module is configured to perform isothermal nucleicacid assembly.
 5. The automated stand-alone multi-module cell editinginstrument of claim 1, wherein the editing module is also a recoverymodule.
 6. The automated stand-alone multi-module cell editinginstrument of claim 1, further comprising a recovery module separatefrom the editing module.
 7. The automated stand-alone multi-module cellediting instrument of claim 1, wherein the growth module measuresoptical density of the growing cells.
 8. The automated stand-alonemulti-module cell editing instrument of claim 7, wherein the growthmodule is configured to measure optical density of growing cellscontinuously.
 9. The automated stand-alone multi-module cell editinginstrument of claim 7, wherein the processor is configured to adjustgrowth conditions in the growth module such that the cells reach atarget optical density at a time requested by a user.
 10. The automatedstand-alone multi-module cell editing instrument of claim 1, wherein thereceptacle configured to receive cells and the one or more receptaclesconfigured to receive nucleic acids are configured to be containedwithin a reagent cartridge.
 11. The automated stand-alone multi-modulecell editing instrument of claim 10, wherein some or all reagentsrequired for cell editing are received by the reagent cartridge.
 12. Theautomated stand-alone multi-module cell editing instrument of claim 11,wherein the reagents contained within the reagent cartridge arelocatable by a script read by the processor.
 13. The automatedstand-alone multi-module cell editing instrument of claim 12, whereinthe reagent cartridge includes reagents and is provided in a kit. 14.The automated stand-alone multi-module cell editing instrument of claim1, wherein the transformation module comprises an electroporationdevice.
 15. The automated stand-alone multi-module cell editinginstrument of claim 14, wherein the electroporation device is aflow-through electroporation device.
 16. The automated stand-alonemulti-module cell editing instrument of claim 1, further comprising afiltration module configured to concentrate the cells and render thecells electrocompetent.
 17. An automated stand-alone multi-module cellediting instrument comprising: a housing configured to house some or allof the modules; a receptacle configured to receive cells; at least onereceptacle configured to receive nucleic acids for desired genomeediting events in the cells; a nucleic acid assembly module configuredto assemble a vector backbone and an editing cassette, wherein thenucleic acid assembly module is configured to accept and assemblenucleic acids to facilitate the desired genome editing events in thecells; a growth module configured to grow the cells; a transformationmodule comprising an electroporator to introduce assembled nucleic acidsinto the cells; a nuclease-directed editing module configured to allowthe assembled nucleic acids to edit nucleic acids in the cells; aprocessor configured to operate the automated multi-module cell editinginstrument based on user input and/or selection of a script; and anautomated liquid handling system to move liquids from the cellreceptacle to the growth module, from the growth module to thetransformation module, and from the transformation module to thenuclease-directed editing module, as well as from the nucleic acidreceptacle to the nucleic acid assembly module, and from the nucleicacid assembly module to the transformation module, all without userintervention.
 18. The automated stand-alone multi-module cell editinginstrument of claim 17, further comprising at least one reagentcartridge containing reagents to perform cell editing in the automatedmulti-module cell editing instrument.
 19. The automated stand-alonemulti-module cell editing instrument of claim 18, wherein thereceptacles for the cells and nucleic acids are disposed within thereagent cartridge.
 20. An automated stand-alone multi-module cellediting instrument comprising: a housing configured to house some or allof the modules; a growth module configured to grow cells; a filtrationmodule configured to concentrate the cells and render the cellselectrocompetent; a transformation module comprising a flow-throughelectroporator to introduce assembled nucleic acids into the cells; acombination recovery and nuclease-directed editing module configured toallow the cells to recover after electroporation in the transformationmodule and to allow the nucleic acids to edit the cells; a processorconfigured to operate the automated multi-module cell editing instrumentbased on user input; and an automated liquid handling system to moveliquids from the growth module to the filtration module, from thefiltration module to the transformation module, and from thetransformation module to the recovery and nuclease-directed editingmodule, without user intervention.